CN1618969A

CN1618969A - Mammalian sytokine-like polypeptide-10

Info

Publication number: CN1618969A
Application number: CNA2004100877931A
Authority: CN
Inventors: D·C·肯克林; B·A·哈尔德曼; A·格罗斯曼
Original assignee: Zymogenetics Inc
Current assignee: Zymogenetics Inc
Priority date: 1997-11-26
Filing date: 1998-11-25
Publication date: 2005-05-25
Also published as: HU227703B1; EP1719780A3; BR9814904A; KR20010032490A; AU1607799A; DK1032671T3; EP1719780A2; IL136076A; WO1999027103A1; EA200401501A1; CN1247780C; EA005581B1; CA2312000A1; DE69824755T2; HUP0100436A2; UA73275C2; ATE269902T1; EP1032671A1; AU739420B2; CZ300849B6

Abstract

A mammalian cytokine-like polypeptide, called Zcyto10, polynucleotides encoding the same, antibodies which specifically bind to the polypeptide, and anti-idiotypic antibodies which bind to the antibodies. Zcyto10 is useful for promoting the healing of wounds and for stimulating the proliferation of platelets.

Description

Mammalian sytokine-like polypeptide-10

The application is to be the dividing an application of Chinese patent application 98812428.9 on November 25th, 1998 applying date.

Background of invention

Cell proliferation and the differentiation of hormone and polypeptide growth factor regulation and control multicellular organism.These can spread molecule exchanges cell mutually and does simultaneously in order to forming cell and organ, and repairs and the regeneration damaged tissue.The example of hormone and somatomedin comprises steroid hormone (as oestrogenic hormon, Testosterone), Rat parathyroid hormone 1-34, follicle stimulating hormone, interleukin-, Thr6 PDGF BB (PDGF), Urogastron (EGF), rHuGM-CSF (GM-CSF), erythropoietin (EPO) and thyrocalcitonin.

Hormone and somatomedin influence cellular metabolism by conjugated protein.Protein can be the conformity membrane albumen relevant with signal transduction path in the cell, such as second messenger system.The protein of other class is shla molecule.

Especially interesting is cytokine, promptly promotes the molecule of cell proliferation and/or differentiation.The example of cytokine comprises erythropoietin (EPO), and it stimulates erythrocytic growth; Thrombopoietin (TPO), its stimulating megakaryocyte pedigree cells whose development; And granulocyte colony-stimulating factor (G-CSF), it stimulates the growth of neutrophilic granulocyte.These cytokines help to recover the anemia patient or accept normal hemocyte level in the cancer chemotherapy patient body.The verified activity in vivo of these cytokines has illustrated the huge clinical potentiality of other cytokine, cytokine stimulant and cytokine antagonist and to their demand.

The invention summary

The present invention satisfies this demand by novel polypeptide and compositions related and method are provided.On the one hand, the invention provides the separation polynucleotide that coding is called as the Mammals four alpha-helix cytokines of Zcyto10.People Zcyto10 polypeptide comprises 176 the amino acid whose sequences that take up the beginning methionine(Met) as shown in SEQ ID NO:1 and SEQ ID NO:2.Think that now amino-acid residue 1-24 is a signal sequence, comprise the aminoacid sequence that the 25th residue (leucine) limited to 176 amino acids residues (L-glutamic acid), by SEQ ID NO:12 and represent ripe Zcyto10 polypeptide.

Another embodiment of the present invention is limited by the sequence of SEQ ID NO:3 and SEQ ID NO:4.The polypeptide of SEQ ID NO:4 comprises 151 amino-acid residues, and wherein the 1-24 amino acids contains signal sequence, and mature sequence comprises that also the 25th amino acids residue (leucine) that is limited by SEQ ID NO:13 is to the 151st amino acids (L-glutamic acid).Another active variant comprises that the 33rd amino acids residue (halfcystine) is to the 176th amino acids residue among the SEQ ID NO:2.This variant is also limited by SEQ ID NO:26.

Mouse Zcyto10 also be comprise such as SEQ ID NO:18 and 19 the polypeptide of 176 amino-acid residues of qualification.Mouse Zcyto10 has the signal sequence that extends to and comprise the 24th amino acids residue glycine from the 1st amino acids residue methionine(Met) of SEQ ID NO:19.Thereby ripe mouse Zcyto10 extends to and comprises the 176th amino acids residue leucine from the 25th amino acids residue leucine of SEQ ID NO:19, is also limited by SEQ ID NO:20.Another active variant is considered to extend to the 176th amino acids from the 33rd amino acids halfcystine of SEQ ID NO:19.This variant is also determined by SEQ ID NO:25.In other embodiments, this polypeptide further comprises affine sign.

A kind of variant of SEQ ID NO:33 and 34 expression mouse Zcyto10.This variant is that 154 amino-acid residues are long, and has the signal sequence that extends to and comprise the 24th amino acids residue glycine from SEQ ID NO:34 the 1st amino acids residue methionine(Met).Thereby mature sequence extends to and comprises the 154th amino acids residue leucine from the 25th amino acids residue leucine of SEQ ID NO:34.Mature sequence is also represented with SEQ ID NO:35.

A second aspect of the present invention provides expression vector, wherein contains (a) transcripting promoter; (b) the DNA section of coding Zcyto10 polypeptide and (c) transcription terminator, wherein promotor, DNA section effectively are connected with terminator.

A third aspect of the present invention provides and has introduced the artificial culture eucaryon or the prokaryotic cell prokaryocyte of expression vector as mentioned above, this DNA section encoded polypeptide of wherein said cell expressing.

Further aspect of the present invention provides basically by the chimeric polyeptides of forming by the first part and the second section of peptide bond connection.The first part of this chimeric polyeptides is made up of following basically: (a) the Zcyto10 polypeptide as shown in SEQ ID NO:2; (b) allelic variant of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:34 or SEQ ID NO:35; (c) with (a) or (b) at least 90% identical protein and peptide.The second section of chimeric polyeptides is made up of another polypeptide such as affine sign basically.In certain embodiment, affine sign is the immunoglobulin Fc polypeptide.The present invention also provides the expression vector and the transfected host cell with the generation chimeric polyeptides of coding chimeric polyeptides.

Another aspect of the present invention provides the antibody of the as above disclosed Zcyto10 polypeptide of energy specific combination, and the antiidiotypic antibody of the anti-Zcyto10 polypeptide antibody that can neutralize.

Another aspect of the present invention provides and has contained and the medicinal pharmaceutical composition of accepting the purifying Zcyto10 polypeptide of carrier associating.Further discuss as this paper institute, in prevention that is characterised in that the disorder that inappropriate cell proliferation, cytodifferentiation or cytokine produce and treatment, such composition can help to regulate cell proliferation, cytodifferentiation or cytokine and produce.More special is that the Zcyto10 polypeptide can be by suppressing prevention and the treatment that cellullar immunologic response helps autoimmune disease.The autoimmune disease of available Zcyto10 treatment comprises IDDM, multiple sclerosis, rheumatoid arthritis or the like.Equally, Zcyto10 polypeptide of the present invention can help anticancer growth or propagation.

But Zcyto10 polypeptide of the present invention also stimulating immune system to resist microorganism or virus infection better.Especially, can systematicness take Zcyto10 to improve individual platelet yield.In addition, Zcyto10 polypeptide of the present invention can be used in tracheae is special or tracheal bronchus the is special application, such as being used for keeping or wound repair of tracheal bronchus epithelium or its basal cell, be used for regulating the sticking cilium removing of mucus generation or residue or being used for asthma, bronchitis or other treatment of diseases of tracheal bronchus road.It also can strengthen wound healing and promote the regeneration of affected tissue, especially helps the treatment of periodontal disease.In addition, the Zcyto10 polypeptide can be used for treating tetter, has substantially such as psoriasis, eczema and xeroderma.

Another embodiment of the present invention relates to the peptide or the polypeptide of the aminoacid sequence of tool Zcyto10 polypeptid belt epi-position part, and this Zcyto10 polypeptide has above-mentioned aminoacid sequence.Although reaching and comprise any length band epitope polypeptide of the whole aminoacid sequence of the invention described above polypeptide also is included among the present invention, but the peptide or the polypeptide of the aminoacid sequence of tool Zcyto10 polypeptid belt of the present invention epi-position part comprise at least 9 of tools, preferably at least 15, more preferably 30-50 amino acid whose polypeptide portion at least.Also claimed is any these polypeptide that merge with another polypeptide or carrier molecule.Such epi-position variant is including, but not limited to SEQ ID NO:25-32.The antibody that partly produces from these band epi-positions of Zcyto10 can be used for purifying Zcyto10 from cell culture medium.

With reference to following detailed description and accompanying drawing, these and other aspect of the present invention will be more obvious.

Detailed Description Of The Invention

All reference that this literary composition is quoted are all quoted as a reference.

Before the present invention is described in detail in detail, defines following term and help understanding it:

Term " affinity labelling " is used herein to finger can be attached to purifying and detection that is beneficial to second peptide species on second peptide species or the polypeptide fragment that the site is provided for second peptide species and combining of substrate.In principle, any peptide or the protein that can obtain antibody or other specific binding agents all can be used as affinity labelling.Affinity labelling comprise poly Histidine fragment, albumin A [people such as Nilsson, EMBO is (1985) J.4:1075; People such as Nilsson, Enzymology method, 198:3 (1991)], glutathione s-transferase [Smith and Johnson, gene 67:31 (1988)], L-glutamic acid-L-glutamic acid affinity labelling [people such as Grussenmeyer, institute of NAS reports 82:7952-4 (1985)], P material, Flag ^TMPeptide [people such as Hopp, biotechnics 6:1204-1210 (1988)], Streptavidin binding peptide or other antigenic epitopes or binding domains.Usually consult people such as Ford, protein expression and purifying 2:95-107 (1991).The coding affinity labelling DNA can buy from the commodity suppliers (as Pharmacia Biotech, Piscataway, NJ).

Term " allelic variant " is used herein to and refers to that a gene occupies any in two or more variations of identical chromosomal foci.Allelic variation is abiogenous by suddenling change, and may cause the phenotypic polymorphism in the population.Transgenation can be the polypeptide that reticent (coded polypeptide no change) or coding have altered aminoacid sequence.Allelic variant one speech also is used in reference to the allelic variant encoded protein matter by gene in this article.

Term " N-terminal " and " C-terminal " are used herein to the position in the expression polypeptide.Allow part in content, these terms are used to represent with reference to a certain polypeptide particular sequence or the degree of approach or relative position partly.For example, certain sequence that is arranged in polypeptide canonical sequence carboxyl terminal promptly is near the canonical sequence C-terminal, but needn't be at the C-terminal of complete polypeptide.

Term " complement/anti-complement to " expression forms non-covalent bonded under proper condition and stablizes right part inequality.For example, vitamin H and avidin (or Streptavidin) are the right typical members of complement/anti-complement.The complement of other example/anti-complement to comprise receptor/ligand to, antibody/antigen (or haptens or epi-position) to, have justice/antisense polynucleotides right, or the like.The dissociated subsequently complement of needs/anti-complement centering, preferably the binding affinity that complement/anti-complement is right is less than 10 ⁹M ^-1

Term " complement of polynucleotide molecule " is the polynucleotide molecule that has with the base sequence of canonical sequence reverse complemental.For example, sequence 5 ' ATGCACGGG 3 ' is a complementary with 5 ' CCCGTGCAT3 '.

The polynucleotide that term " contiguous nucleotide sequence " expression has identical with another polynucleotide or complementary contiguous sequence.Contiguous sequence is called as one section whole or these polynucleotide part " overlapping " with the polynucleotide sequence of giving.For example, polynucleotide sequence 5 '-the representative contiguous nucleotide sequence of ATGGCTTAGCTT-3 ' is 5 '-TAGCTTgagtct-3 ' and 3 '-gtcgacTACCGA-5 '.

Term " degenerate core nucleotide sequence " expression comprises the nucleotide sequence of one or more degenerate codons (with comparing with reference to polynucleotide molecule of coded polypeptide).Degenerate codon comprises different nucleotide triplets, but the identical amino-acid residue of encoding (that is, GAU and GAC triplet all encode aspartic acid).

Term " expression vector " is used to represent to contain and the additional clip segmental linearity of effective purpose of connecting peptide coding or the ring-shaped DNA molecule that are used to transcribe.These additional clip comprise promotor and terminator sequence, also can comprise one or more replication orgin, one or more selective marker, enhanser, polyadenylation signal, or the like.Expression vector derives from plasmid or viral DNA usually, or both compositions of possibility all comprise.

Therefore term " separation " is meant that polynucleotide molecule breaks away from from natural genotypic environment when being used for polynucleotide molecule, and removed other unnecessary or undesired encoding sequence, is in a kind of form that is applicable to genetically engineered protein production system.Such isolated molecule is those molecules that break away from its natural surroundings, comprises cDNA and genomic clone.DNA isolation molecule of the present invention does not have other usually relevant gene, but can comprise naturally occurring 5 ' and 3 ' untranslated region, such as promotor and terminator.The evaluation of relevant range is tangible [for example consulting Dynan and Tijan, natural 316:774-78 (1985)] to this area routine techniques personnel.

The polypeptide of " separation " or protein are polypeptide and the protein under the condition that is in outside its natural surroundings, for example isolated polypeptide or protein from blood and animal tissues.In a preferred form, other polypeptide of essentially no other polypeptide, especially animal-origin of isolated polypeptide.The polypeptide of high purified form preferably is provided, and promptly purity surpasses 95%, and more preferably purity surpasses 99%.When being applied to herein, term " isolating " is not got rid of the phase homopolypeptide and is existed with other physical form, as dimer or glycosylated or deutero-form.

Term " effectively connect " is when referring to dna fragmentation, and the expression fragment is suitably arranged so that its function is consistent with purpose, as transcription initiation in promotor and run through encoding sequence and proceed to terminator.

Term " normal homologue (ortholog) " expression derives from species, is the polypeptide or the protein of the polypeptide of another species or proteinic function counterpart.Sequence difference between the normal homologue is the result that species form.

" secondary homologue (parolog) " is by a kind of mutual difference of organism generation but the protein of structurally associated.Secondary homologue is considered to produce by gene redundancy.The secondary each other homologue of α-Zhu Danbai, beta-globin and myohaemoglobin for example.

" polynucleotide " are for reading to the deoxyribonucleotide of 3 ' end or the strand or the double-stranded polymer of ribonucleotide base from 5 ' end.Polynucleotide comprise RNA and DNA, and are separable from natural origin, can externally synthesize, or in conjunction with natural molecule and synthetic molecules and make.The size of polynucleotide is expressed as base pair (abbreviation " bp "), Nucleotide (" nt ") or kilobase (" kb ").As long as content allows, strand or double-stranded polynucleotide can be described in latter two term.When this term is used in reference to duplex molecule, expression be total length, be interpreted as being equal to term " base pair ".Those skilled in that art will recognize that two chain lengths of double-stranded polynucleotide may be slightly different, and its end may be cut because of enzyme and form the stagger shape; Thereby all Nucleotide in the double-stranded polynucleotide molecule not necessarily match.These unpaired tip lengths generally are no more than 20nt.

" polypeptide " is amino-acid residue polymer natural generation or that synthetic connects with peptide bond.The polypeptide of not enough about 10 amino-acid residues is commonly referred to " peptide ".

With its art-recognized implication, expression contains can be in conjunction with the Gene Partial of RNA polymerase and initial dna sequence dna of transcribing at this for term " promotor ".Normal 5 ' the non-coding region of finding to be positioned at gene of promoter sequence, but not always not like this.

" protein " is the macromole that comprises one or more polypeptide chain.Protein also can contain non-peptide component, as glycosyl.Sugar and other non-peptide substituting group can be added on the protein by producing this proteinic cell, and have nothing in common with each other in different cell types.Protein is represented with its amino acid backbone structure at this paper; Substituting group such as glycosyl do not specialize usually, but still may exist.

Term " acceptor " expression can combine and mediate part and act on cell associated protein on the cell with bioactive molecules (being part).Membrane-bound receptor is characterised in that the structure of Multidomain, comprises the outer ligand binding domains of born of the same parents and relates generally to born of the same parents' internal effect minor structure territory of signal transduction.Part and receptors bind have caused the conformation transition of acceptor, thereby cause other intermolecular interaction in effector structural domain and the cell.This interaction causes the change of cellular metabolism successively.Comprise the moving of transfer, film fat, cell adhesion, the hydrolysis of inositol fat and the hydrolysis of phosphatide of raising, the cell calcium of genetic transcription, phosphorylation, dephosphorylation, cyclic amp output with the related Metabolic activity of receptor-ligand binding.In general, acceptor can be membrane-bound, kytoplasm or nuclear; Monomeric (as thyrotropin receptor, B-adrenergic receptor) or polymeric (as pdgf receptor, growth hormone receptor, IL-3 acceptor, GM-CSF acceptor, G-CSF acceptor, erythropoietin receptor and IL-6 acceptor).

The dna sequence dna of term " secretory signal sequence " presentation code polypeptide (" secretion peptide "), described polypeptide can instruct this bigger polypeptide to pass the Secretory Pathway of the cell that synthesizes it as the component of a bigger polypeptide.This bigger polypeptide is cleaved usually to remove the secretion peptide in by the process of Secretory Pathway transhipment.

Term " splice variant " is used herein to all interchangeable form of the RNA that expression come out by genetic transcription.Splice variant is the natural generation by the interchangeable splice site that utilizes transcribe rna intramolecularly (or being between the RNA molecule of transcribing respectively under the less situation) generally, and can cause by good several mRNA of same genetic transcription.The splice variant codified contains the different aminoacids polypeptide of sequence.The term splice variant splice variant encoded protein matter of the mRNA that also represents in this article to come out by genetic transcription.

Multimeric molecule amount and the length measured with coarse analytical procedure (as, gel electrophoresis) are interpreted as approximation.When this value is expressed as " approximately " X or " being similar to " X, said X value is interpreted as being accurate to ± and 10%.

Conserved amino acid among the spiral D of Zcyto10 can be used as the instrument of differentiating the new family member.Spiral D is that topnotch is conservative, has about 32% homogeny with the spiral D of IL-10.For example, reverse transcriptase polymerase chain reaction (RT-PCR) can be used for the sequence of amplification coding available from the RNA conserved regions (above structural domain, zone or primitive) of all tissue-derived or clone.Especially, design can be used for this purpose from the height degenerated primer of Zcyto10 sequence.

In a preferred embodiment of the invention, isolating polynucleotide can be hybridized to the similar size area of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:18, SEQ ID NO:33 or its complementary sequence under rigorous condition.In general, rigorous condition is chosen as under ionic strength of determining and pH than low about 5 ℃ of the heat fusion joint (Tm) of this distinguished sequence.Tm is 50% target sequence and the temperature of mating probe hybridization fully (under ionic strength of determining and pH).General rigorous condition be about 0.02M of salt concn or pH be lower than 7 and temperature at least about 60 ℃.As previously mentioned, separation polynucleotide of the present invention comprise DNA and RNA.The method that is used for DNA isolation and RNA is well known in the art.Separate people such as to prepare total RNA[Chirgwin, biological chemistry 18:52-94, (1979) with caesium chloride density gradient centrifugation subsequently with the Guanidinium hydrochloride extraction].Poly (A) ⁺RNA can use Aviv and Leder, and institute of NAS reports the method preparation of 69:1408-1412 (1972) from total RNA.The available currently known methods preparation of complementary DNA (cDNA) is from poly (A) ⁺RNA.Available then for example hybridization or PCR differentiate and the polynucleotide that separate coding Zcyto10 polypeptide.

In addition, polynucleotide of the present invention can be synthetic with dna synthesizer.Usually institute's choosing method is the phosphoramidite method.If the double-stranded DNA of application need chemosynthesis prepares each complementary strand so respectively such as gene or gene fragment are synthetic.The production of short gene (60-80bp) is simple technically, can then its annealing be finished by synthetic complementary strand.Yet (＞300bp) production must be carried out special strategy, because each round-robin coupling efficiency seldom can reach 100% in the DNA chemosynthesis for longer gene.In order to overcome this problem, synthetic gene (two strands) is that the single-chain fragment of 20-100 Nucleotide is assembled into modular form by length.Consult Glick, Bernard R. and Jack J.Pasternak, molecular biotechnology is learned, the principle of recombinant DNA and application (ASM press, Washington D.C., 1994), Itakura, people such as K, the synthetic and purposes of synthetic oligonucleotide, biological chemistry yearbook (Annu.Rev.Biochem.) 53:323-356 (1984), and Climie, S. wait the people, the chemosynthesis of thymidylate synthase gene, institute of NAS reports 87:633-637 (1990).

Those skilled in the art will recognize that, be disclosed in SEQ ID NO:1, the sequence in 2,3 and 4 has been represented two allelotrope of people, and SEQ ID NO:18,19,33 and 34 have represented two allelotrope of mouse.Can survey other allelic variant of cloning these sequences from the cDNA of Different Individual or genomic library by standard step.Can be by standard step by surveying the allelic variant of cloning this sequence from the cDNA or the genomic library of Different Individual.The allelic variant of dna sequence dna shown in the SEQ IDNO:1 comprises those variants that contain silent mutation and the sudden change variant that causes aminoacid sequence to change wherein, all within the scope of the present invention, and as the protein of SEQ IDNO:2 allelic variant too.Generation from the reservation of alternate manner montage mRNA the cDNA of Zcyto10 polypeptide characteristic, also be included in the scope of the invention, too by these cDNA and mRNA encoded polypeptide.By surveying cDNA or genomic library, can clone the allelic variant and the splice variant of these sequences by standard step known in the art from Different Individual or tissue.

Respective egg white matter and polynucleotide (" species normal homologue ") have been the present invention further provides from other species.Interested especially at present is from other mammiferous Zcyto10 polypeptide, comprises Zcyto10 polypeptide mouse, pig, sheep, ox, dog, cat, horse and other primates.The proteinic species normal homologue of Zcyto10 of information provided by the invention and composition and conventional clone technology can being joined together to clone people.For example, available available from the mRNA clone cDNA that expresses this proteinic tissue or cell type.The available probe of pressing the open sequences Design of this paper is identified the suitable source of mRNA by surveying the Northern trace.MRNA from positive tissue or clone prepares the library subsequently.Can in all sorts of ways then and separate the cDNA of coded protein, as personnel selection or mouse is whole or Partial cDNA is surveyed, or use based on one or more sets degeneracy probes of open sequence and survey.Also available polymerase chain reaction of cDNA or PCR people such as (, United States Patent (USP) the 4th, 683, No. 202) Mullis method is used by the primer of the open sequences Design of this paper and is cloned.In other method, can utilize the cDNA library to transform or transfection host cell, and with the expression of this proteinic antibody test purpose cDNA.Similar technique also can be used in the separation of genomic clone.As using and require patent protection; " the separation polynucleotide of coded polypeptide, said polynucleotide are by SEQ ID NO:2,4,12,13,19,20,25,26,34 and 35 expressions " this word means all allelic variants and the species normal homologue that comprises these polypeptide.

The present invention also provides protein and peptide and the essentially identical isolated protein polypeptide of its species normal homologue with SEQ ID NO:2." isolating " means protein or the polypeptide under the condition that is present in outside its natural surroundings, as breaking away from autoblood and animal tissues.In a preferred form, other polypeptide of isolated polypeptide essentially no other polypeptide, especially animal-origin.The polypeptide that preferably provides is highly purified form, and promptly purity is higher than 95%, and more preferably purity surpasses 99%." basic identical " used herein speech means polypeptide and sequence shown in the SEQ ID NO:2,4,12,13,19,20,25,26,34 and 35 or its species normal homologue to be had 50%, preferred 60%, more preferably at least 80% identical.Such polypeptide preferably with SEQ ID NO:2 or its species normal homologue have at least 90% identical, most preferably 95% or more how identical.Sequence homogeny percentage can be measured with ordinary method.Consult, for example, people such as Altschul, Bull.Math.Bio.48:603-616 (1986) and Henikoff and Henikoff, institute of NAS reports 89:10915-10919 (1992).Speak briefly, exactly with the open point penalty of breach be 10, to extend point penalty be 1 and " blossom62 " rating matrix of (amino acid is represented with the standard one-letter symbol) as shown in table 1 Henikoff and Henikoff (the same) to breach, two aminoacid sequences are compared, so that the comparison scoring is best.The homogeny percentage is calculated as follows:

Table 1

A R N D C Q E G H I L K M F P S T W Y V

A 4

R -1 5

N -2 0 6

D -2 -2 1 6

C 0 -3 -3 -3 9

Q -1 1 0 0 -3 5

E -1 0 0 2 -4 2 5

G 0 -2 0 -1 -3 -2 -2 6

H -2 0 1 -1 -3 0 0 -2 8

I -1 -3 -3 -3 -1 -3 -3 -4 -3 4

L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4

K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5

M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5

F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6

P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7

S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4

T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5

W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11

Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7

V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4

Utilize ratio as disclosed above by the sequence homogeny between similarity method mensuration polynucleotide molecule.

Variant Zcyto10 polypeptide or essentially identical protein and peptide characteristic are the one or more aminoacid replacement of tool, deletion or interpolation.These change the change of preferred less character, promptly conserved amino acid replace (seeing Table 2) and other can remarkably influenced protein or polypeptide fold or active replacement; Little deletion, generally about 1-30 amino acid whose deletion; And little amino or carboxyl terminal extension, such as the aminoterminal methionine residues, up to the long little connection peptides of about 20-25 residue, or be beneficial to the little extension (affinity labelling) of purifying, such as poly Histidine fragment, albumin A [people such as Nilsson, EMBO is (1985) J.4:1075; People such as Nilsson, Enzymology method, 198:3 (1991)], glutathione S shifts alcohol [Smith and Johnson, gene 67:31 (1988)], or other antigenic epitopes or binding domains.Usually consult people such as Ford, protein expression and purifying 2:95-107 (1991).The DNA of coding affinity labelling can be available from the goods providers place (as, Pharmacia Biotech, Piscataway, NJ).

Table 2

Conserved amino acid replaces

Alkalescence: arginine

Methionin

Histidine

Tart: L-glutamic acid

Aspartic acid

Polar: glutamine

L-asparagine

Hydrophobic: leucine

Isoleucine

Xie Ansuan

Aromatic: phenylalanine

Tryptophane

Tyrosine

Little: glycine

L-Ala

Serine

Threonine

Methionine(Met)

Various other polypeptide syzygys relevant multimerization protein of one or more polypeptide syzygys (and contain) have been the present invention further provides.For example, can be by United States Patent (USP) the 5th, 155,027 and 5,567, No. 584 disclosed is prepared into syzygy with dimerizing protein with the Zcyto10 polypeptide.Comprise the constant region for immunoglobulin structural domain at the preferred dimerizing protein aspect this.Immunoglobulin (Ig)-Zcyto10 polypeptide syzygy can be expressed (to produce various polymer Zcyto10 analogues) in genetically engineered cell.The supplementary structure territory can be merged to the Zcyto10 polypeptide, they are oriented to specific cell, tissue or macromole (as collagen).For example, merge, Zcyto10 polypeptide or protein can be oriented to predetermined cell type by part with polypeptide and specific combination target cell surface receptor.In this way, polypeptide and protein orientation can be used for the treatment of or diagnostic purpose.Can be with Zcyto10 polypeptide and two or more meromixis, such as affinity labelling that is used for purifying and oriented structure territory.The polypeptide syzygy also can comprise one or more cleavage sites, especially the cleavage site between structural domain.Consult people such as Tuan, reticular tissue research (Connective Tissue Research) 34:1-9 (1996).

Protein of the present invention can also comprise the amino-acid residue that non-natural exists.The amino acid that non-natural exists comprises and is not limited to trans-3-methylproline, 2,4-methylene proline(Pro), cis-4-Hydroxyproline, trans-4-Hydroxyproline, sarcosine, allothreonine, methylthreonine, the hydroxyethyl halfcystine, the hydroxyethyl homocysteine, the nitro glutamine, high glutamine, pipecolic acid, the thiazolidine carboxylic acid, dehydroproline, 3-and 4-methylproline, 3,3-dimethyl proline(Pro), Terleu, norvaline, 2-pyridine L-Ala (2-azaphenylalanine), 3-pyridine L-Ala, 4-pyridine L-Ala and 4-fluorophenylalanine.Several method known in the art can be used for existing amino-acid residue to mix protein non-natural.For example, can utilize a vitro system, wherein nonsense mutation all suppresses sub-tRNA with chemical aminoacylation and suppresses.The method of synthesizing amino acid and aminoacyl-tRNA is known in the art.

Indispensable amino acid in the polypeptide of the present invention can be identified [Cunningham and Wells, science 244:1081-1085 (1989) as site-directed mutagenesis or alanine scanning mutagenesis by the method known in the art; People such as Bass, institute of NAS report 88:4498-4502 (1991)].In one technology of back, each the residue place in molecule imports single alanine mutation, and the biologic activity (as aglucon combination and signal transduction) of check gained mutating molecule is to identify the amino-acid residue relatively more crucial to molecular activity.Also can measure the site of part-protein interaction by the crystalline structure analysis that the technology such as nucleus magnetic resonance, crystallography technology or photoaffinity labeling is determined.Consult, for example, people such as de Vos, science 255:306-312 (1992); Smith etc., molecular biology magazine 224:899-904 (1992); Wlodaver etc., FEBS Lett.309:59-64 (1992).Indispensable amino acid also can be from inferring with the homology analysis of related protein.

Available known mutagenesis and screening method preparation are also checked multiple aminoacid replacement, as Reidhaar-Olson and disclosed method (the science 241:53-57 of Sauer, 1988) or Bowie and the disclosed method of Sauer (institute of NAS reports 86:2152-2156,1989).Briefly, these author's disclosed methods are the two or more positions randomizations that make simultaneously in the polypeptide, and the selection function polypeptide checks order then and allows the replacement scope through the polypeptide of mutagenesis with what determine each position.Other available method comprises that phage display is (as Lowman etc., biological chemistry 30:10832-10837 (1991); Ladner etc., U.S. Patent number 5,223,409; Huse, the open WO92/06204 of WIPO) and regional directed mutagenesis (Derbyshire etc., gene 46:145,1986; Ner etc., DNA7:127,1988).

Mutafacient system as disclosed above can be in conjunction with the heavy body screening method to detect the mutagenesis activity of proteins of cloning in the host cell.Preferred in this test comprise cell proliferation test and based on the part of biosensor in conjunction with test, below these tests are had description.The mutagenized dna molecule of coding activated protein or its part (as the part binding fragment) can reclaim from host cell and check order rapidly with modem devices.But the importance of each amino-acid residue in these method rapid determination desired polypeptides, and applicable to the polypeptide of unknown structure.

Use aforesaid method, this area routine techniques personnel can prepare and SEQ ID NO:2,4,12,13,19,20,25,26,34 and 35 or its allelic variant is basic identical and keep the kind peptide species of wild-type protein characteristic.Expressed and claim that " polypeptide that SEQ ID NO:2 represents " comprise all allelic variants and the species normal homologue of this polypeptide as this paper.

Protein polypeptide of the present invention, comprise full length protein, protein fragments (as the part binding fragment) and fusion polypeptide all routinely technology in genetically engineered host cell, produce.Proper host cell is that those can be transformed by foreign DNA or transfection and cell type that can incubation growth, comprises the higher eucaryotic cells of bacterium, fungal cell and cultivation.The culturing cell of preferred eukaryotic cell, especially multicellular organism.Operation cloned DNA molecule and the technology that foreign DNA is introduced various host cells is disclosed in the following document: people such as Sambrook, molecular cloning: laboratory manual, second edition (press of cold spring harbor laboratory, the cold spring port, New York, 1989), reach people such as Ausubel, the same.

Polynucleotide of the present invention are generally the cDNA sequence, the coding aforementioned polypeptides.The dna sequence dna of code book invention polypeptide is made up of a series of codons, and each amino-acid residue of polypeptide is encoded by codon, and each codon is made up of 3 Nucleotide.

Amino-acid residue is by following corresponding codon coding.

L-Ala (Ala) is by GCA, GCC, GCG or GCT coding;

Halfcystine (Cys) is by TGC or TGT coding;

Aspartic acid (Asp) is by GAC or GAT coding;

L-glutamic acid (Glu) is by GAA or GAG coding;

Phenylalanine (Phe) is by TTC or TTT coding;

Glycine (G1y) is by GGA, GGC, GGG or GGT coding;

Histidine (His) is by CAC or CAT coding;

Isoleucine is by ATA, ATC or ATT coding;

Methionin (Lys) is by AAA or AAG coding;

Leucine (Leu) is by TTA, TTG, CTA, CTC, CTG or CTT coding;

Methionine(Met) (Met) is encoded by ATG;

L-asparagine (Asn) is by AAC or AAT coding;

Proline(Pro) (Pro) is by CCA, CCC, CCG or CCT coding;

Glutamine (Gln) is by CAA or CAG coding;

Arginine (Arg) is by AGA, AGG, CGA, CGC, CGG or CGT coding;

Serine (Ser) is by AGC, AGT, TCA, TCC, TCG or TCT coding;

Threonine (Thr) is by ACA, ACC, ACG or ACT coding;

Xie Ansuan (Val) is by GTA, GTC, GTG or GTT coding;

Tryptophane (Trp) is encoded by TGG; And

Tyrosine (Tyr) is by TAC or TAT coding.

Can recognize that according to the present invention when cDNA required patent protection as mentioned above, what be interpreted as requiring patent protection was sense strand, antisense strand and justice is arranged and double-stranded DNA that antisense strand is annealed together and formed by hydrogen bond separately.Require the messenger RNA(mRNA) (mRNA) that also has code book invention polypeptide of patent protection to reach by the coded mRNA of above-mentioned cDNA.Messenger RNA(mRNA) (mRNA) is used and the sub-coded polypeptide of same password of above indicating, and just each thymidine (T) is all replaced by uridine (U).

Usually, in expression vector, other required genetic elements of the dna sequence dna of coding Zcyto7 polypeptide and its expression effectively is connected, and described element generally comprises transcripting promoter and terminator.Although those skilled in the art can think that selective marker can be provided on the carrier of separating in some system; and duplicating of foreign DNA can provide by being integrated into the host cell gene group, but carrier also comprises one or more selective markers and one or more replication orgin usually.Promotor, terminator, selective marker, carrier and other selection of components are the conventional design problems within those of ordinary skills' level.Many these elements all have description in the literature, and can be available from the goods providers place.

In order to instruct the Zcyto10 polypeptide to enter the Secretory Pathway of host cell, can in expression vector, provide secretory signal sequence (also claiming leader sequence, preceding former sequence or presequence).Secretory signal sequence can be this protein itself, or can be from another secretory protein (as t-PA) or de novo synthesis.Secretory signal sequence is connected with the Zcyto10 dna sequence dna with the proper reading frame frame.Although some signal sequence can be arranged in the target DNA sequence other position (consult, as people such as Welch, United States Patent (USP) the 5th, 037,743; People such as Holland, United States Patent (USP) the 5th, 143,830), but secretory signal sequence is usually located at 5 ' end of the dna sequence dna of coding desired polypeptides.

The method that foreign DNA is introduced the mammals host cell comprises transfection [people such as Wigler, the cell 14:725 (1978) of calcium phosphate mediation; Corsaro and Pearson, somatic cell genetics 7:603,1981; Graham and Van der Eb, virusology 52:456 (1973)], electroporation people such as [, EMBO is (1982) J.1:841-845] Neumann, [people such as Ausubel compiles in the transfection of DEAE-dextran mediation, the general handbook of molecular biology (John Wiley and Sons, Inc., NY, 1987)], and liposome-mediated transfection [people such as Hawley-Nelson, Focus 15:73 (1993); People such as Ciccarone, Focus 15:80 (1993)].Production of recombinant polypeptides is disclosed in the following document in the cells of mamma animals of cultivating: people such as Levinson, United States Patent (USP) the 4th, 713, No. 339; People such as Hagen, U.S. Patent number 4,784,950; People such as Palmiter, U.S. Patent number 4,579,821; And Ringold, U.S. Patent number 4,656,134.Suitable cultivation cells of mamma animals comprises COS-1 (ATCC NO.CRL1650), COS-7 (ATCC NO.CRL1651), BHK (ATCC NO.CRL1632), BHK570 (ATCC NO.CRL10314), 293[ATCC NO.CRL1573; People such as Graham, general virology magazine (J.Gen.Virol) 36:59-72 (1977)] and Chinese hamster ovary cell (as CHO-K1; ATCC NO.CCL61) clone.Suitable clone in addition is well known in the art, and can be available from public depositary institution, as American type culture collection, and Rockefeller, the Maryland State.In general, preferred strong transcripting promoter is as the promotor from SV-40 or cytomegalovirus.Consult as, U.S. Patent number 4,956,288.Other suitable promotor comprises promotor (U.S. Patent number 4,579,821 and 4,601,978) and the adenovirus major late promoter from metallothionein gene.

Usually the cultivation cells of mamma animals that utilizes medicament selection to select to insert foreign DNA.These cells are commonly referred to " transfectant ".The cell of having cultivated and goal gene can have been passed to filial generation under the condition that has selective agent to exist is called " stable transfectant ".Preferred selective marker is the gene of coding to the resistance of antibiotic neomycin.Be chosen under the condition that Xin Meisu type medicine (as G418 or the like) exists and carry out.Selective system also can be used for improving the expression level of goal gene, and this process is called " amplification ".Amplification is undertaken by following process: cultivate transfectant under the condition that low-level selective agent exists, the amount that increases selective agent then produces the cell that high level is introduced the product of gene to select.The selective marker that preferably can increase is a Tetrahydrofolate dehydrogenase, and it gives the resistance to methotrexate.Other drug resistance gene (as Totomycin resistance, wide spectrum resistance, tetracycline Transacetylase) also can use.Import other mark that changes phenotype, as green fluorescent protein, or cell surface protein (as CD4, CD8, MHC I quasi-molecule), P-ALP all can be used to select transfectional cell by the method for FACS sorting art or magnetic bead isolation technique and so on from non-transfected cells.

Other higher eucaryotic cells also can be used as the host, comprises insect cell, vegetable cell and avian cell.The conversion of insect cell and the production of middle allogenic polypeptide thereof have been disclosed in people's such as people's such as Guarino United States Patent (USP) the 5th, 162,222, Bang the United States Patent (USP) the 4th, 775,624 and the open WO94/06463 of WIPO.Make carrier expressing gene in vegetable cell with Agrobacterium rhizogenes and done review [bio-science magazine (J.Biosci.) is 11:47-58 (1987) (Bangalore)] by people such as Sinkar.Insect cell can use baculovirus recombinant chou [usually derived from autographa california nuclear polyhedrosis virus (AcNPV)] to infect.See King, L.A. and Possee, R.D., baculovirus expression system: lab guide (Chapman﹠amp; Hall, London); O ' Reilly, people such as D.R., rhabdovirus expression vector: laboratory manual (Oxford University Press, New York, 1994); And Richardson, C.D. edits, baculovirus expression scheme, molecular biology method (Humana press, Totowa, NJ, 1995).Second kind of method for preparing reorganization Zcyto10 baculovirus used the system based on transposon, and this systematic review is in Luckow, and V.A. waits the people, and Journal of Virology 67:4566-79 is in 1993.This system that has used transfer vector is at Bac-to-Bac ^TMTest kit (LifeTechnologies, Rockyille, on sale in MD).This system's utilization contains the transfer vector pFastBacl of Tn7 transposon ^TM(Life Technologies) is transferred to as the big plasmid that is called as " rod granule " with the DNA of the Zcyto10 polypeptide of will encoding and remains in the baculovirus genome in the intestinal bacteria.See Hill-Perkins, M.S. and Possee, R.D., general virology magazine 71:971-6, (1990); Bonning, people such as B.C., general virology magazine 75:1551-6 (1994); And Chazenbalk, G.D. and Rapoport, B., journal of biological chemistry 270:1543-9 (1995).In addition, transfer vector can be included in the C-of expressed Zcyto10 polypeptide or the frame endomixis body [Grussenmeyer that the N-end has merged the coding DNA of epi-position mark (as Glu-Glu epi-position mark), T. wait the people, institute of NAS reports 82:7952-4 (1985)].The transfer vector conversion that contains Zcyto10 can be entered intestinal bacteria with technology known in the art, and filter out the rod granule that contains the interruption LacZ gene that is designated as recombinant baculovirus.Contain the genomic bacmid dna of recombinant baculovirus and be used for transfection fall army worm cell (as the Sf9 cell) with the routine techniques separation.The recombinant virus of expressing Zcyto10 can produce.Prepare the recombinant virus original seed with this area common method.

Recombinant virus can be used for host cells infected, normally derived from the clone of autumn mythimna separata (fall army worm).Always see, Glick and Pasternak, molecular biotechnology is learned: the principle of recombinant DNA and application, ASM press, Washington D.C. (1994).Another suitable clone is the High Five O derived from cabbage looper ^TMClone (Invitrogen) (U.S. Patent number 5,300,435).The serum free medium that the manufacturer provides can be used for cultivating and keeping cell.Suitable medium is Sf900 II for the Sf9 cell ^TM(Life Technologies) or ESF 921 ^TM(Expression Systems); For the cabbage looper cell is Ex-Cell0405 ^TM(JRH Biosciences, Lenexa, KS) or Express Five O ^TM(LifeTechnologies).Cell is from being about 2-5 * 10 ⁵The inoculum density of individual cell grows to 1-2 * 10 ⁶During individual cell density, add the recombinant virus original seed with the infection multiplicity (MOI) of 0.1 to 10 (being more typically nearly 3).Program thereby often be described in the laboratory manual (King, L.A. and Possee, R.D., the same; O ' Reilly, D.R. etc., the same; Richardson, C.D., the same).Available subsequently methods described herein are purified into the Zcyto10 polypeptide from supernatant.

The fungal cell comprises yeast cell, and especially the cell of yeast belong also can be used among the present invention, as is used to produce protein fragments or fusion polypeptide.The method that also therefrom produces recombinant polypeptide with the foreign DNA transformed yeast cell is disclosed in following file, for example, and Kawasaki, U.S. Patent number 4,599,311; Kawasaki etc., U.S. Patent number 4,931,373; Brake, U.S. Patent number 4,870,008; Welch etc., U.S. Patent number 5,037,743; With Murray etc., U.S. Patent number 4,845,075.By the phenotype of being determined by selective marker, normally the drug resistance or the ability of growing under the condition that lacks certain special nutrient (as leucine) are selected transformant.The preferred vector system that is used for yeast is the POT1 carrier system, is disclosed in people's such as Kawasaki the United States Patent (USP) the 4th, 931,373, and this system can be by selecting transformant containing on the substratum of glucose growth.Be used for the suitable promotor of yeast and terminator comprise from the glycolysis-gene (see, as, Kawasaki, U.S. Patent number 4,599,311; People such as Kingsman, U.S. Patent number 4,615,974; And Bitter, U.S. Patent number 4,977,092) and the promotor and the terminator of alcohol dehydrogenase gene.Also can consult United States Patent (USP) the 4th, 990, No. 446; The 5th, 063, No. 154; The 5th, 139, No. 936 and the 4th, 661, No. 454.Other zymic conversion system that is used for known in the art, described yeast comprises: multiple-shaped nuohan inferior yeast, schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, pichia pastoris phaff, pichia methanolica (Pichiamethanolica), Pichia guilliermondii and maltose candiyeast (Candidamaltosa).Consult, as Gleeson etc., general microbiology magazine 132:3459-3465 (1986) and Cregg, United States Patent (USP) the 4th, 882, No. 279.Can utilize the Aspergillus cell according to the method for No. the 4th, 935,349, the United States Patent (USP) of McKnight etc.The method of transform producing yellow branch top spore (Acremonium chrysogenum) is disclosed in people's such as Sumino No. the 5th, 162,228, the United States Patent (USP).The method that transforms Neurospora is disclosed in No. the 4th, 486,533, the United States Patent (USP) of Lambowitz.

Be described among WIPO open WO97/17450, WO97/17451, WO98/02536 and the WO98/02565 as the method that the host produces recombinant protein with pichia methanolica.The dna molecular that is used to transform pichia methanolica will be made the double-stranded circular plasmid usually, preferably first linearizing before transforming.In order to produce polypeptide in pichia methanolica, promotor in the preferred plasmid and terminator are gene in the pichia methanolica, utilize promotor and the terminator of gene (AUG1 or AUG2) as pichia methanolica alcohol.Other useful promotor comprises constructed by dihydroxy acetone synthetase (DHAS) gene, the promotor of hydrogenlyase (FMD) gene and catalase (CAT) gene.Be integrated into host chromosome for the ease of DNA, the whole expression fragment both sides of preferred plasmid are the host DNA sequence.The selective marker that is preferred for pichia methanolica is a pichia methanolica ADE2 gene, its Phosphoribosyl-5-aminooimidazole carboxylase (AIRC that encodes; EC4.1.1.21), this enzyme can make the ade2 host cell grow when lacking VITAMIN B4.In order to wish few mass industrialized production, preferably use wherein two host cells that methyl alcohol utilizes gene (AUG1 and AUG2) all to lack with methyl alcohol.In order to produce secretary protein, the preferred defective host cell of vacuole protein enzyme gene (PEP4 and PRB1).Can utilize electroporation to promote the plasmid that contains the desired polypeptides coding DNA to introduce the pichia methanolica cell.Preferably transform the pichia methanolica cell with electroporation method, used electric field is the pulsed electrical field of exponential decay, and strength of electric field is 2.5-4.5kV/cm, preferably is about 3.75kV/cm, and time constant (τ) is the 1-40 millisecond, most preferably from about 20 milliseconds.

Prokaryotic host cell comprises that the bacterial strain of intestinal bacteria, bacillus and other genus also can be used as host cell of the present invention.The technology that transforms these hosts and cloning by expression foreign DNA wherein is well-known in this area (seeing, as, Sambrook etc., the same).When expressing the Zcyto10 polypeptide in the bacterium such as intestinal bacteria, this polypeptide can be retained in the tenuigenin and (be generally insoluble particles), or can be guided in the periplasmic space by the bacterium secretion sequence.In the previous case,, reclaim particle and also use as guanidinium isothiocyanate or urea-denatured with lysis.Can as urea soln being dialysed and being used in combination reduced form and Sleep-promoting factor B, to the buffer salt solution dialysis, make sex change polypeptide folding and dimerization again again by the dilution denaturing agent then.In the later case, polypeptide can have functional form to reclaim with solubility from periplasmic space, promptly by smudge cells (as by supersound process or osmotic shock) with the content that discharges periplasmic space and reclaim protein and finish, like this can sex change and fold again.

Transform or cells transfected is incubated in the substratum that contains the essential nutrition of selected host cell growth and other composition according to conventional procedure.Various suitable medium comprise known composition substratum and complex medium, all are known in the art, and generally include carbon source, nitrogenous source, indispensable amino acid, VITAMIN and mineral substance.If desired, also can comprise component such as somatomedin or serum in the substratum.Substratum will or lack certain by for example medicament selection usually must nutrient select to contain the cell that external source imports DNA, and this essential nutrient that lacks can be replenished by the selective marker of carrying on the expression vector or cotransfection enters host cell.About 25 ℃-35 ℃ with the pichia methanolica cell cultures in the substratum that contains enough carbon sources, nitrogenous source and micro-nutrient.In liquid culture, provide enough air flows with ordinary method, such as shaking the spraying of bottle or fermentor tank for a short time.The preferred culture medium of pichia methanolica is YEPD (2%D-glucose, 2%Bacto ^TMPeptone (the Difco laboratory, the Detroit, MI), 1%Bacto ^TMYeast extract (Difco laboratory), O.004% VITAMIN B4 and leucine O.006%L-).

In one aspect of the invention, produce new protein by culturing cell, this cell is used to screen this proteinic one or more acceptors, comprises natural receptor, and the agonist of natural aglucon and antagonist.

Protein separation:

Preferred purifying polypeptide of the present invention is to being not less than 80% purity, more preferably be not less than 90% purity, even more preferably be not less than 95% purity, especially preferred purity pharmaceutically, promptly with respect to the impurity macromole, particularly other protein and nucleic acid, purity surpasses 99.9%, and does not contain infectious agent and pyrexin.Preferably, purified polypeptide does not contain other polypeptide substantially, particularly other animal source polypeptide.

Recombinant polypeptide (or chimeric polyeptides) available classification separation of expressing and/or conventional purification process and medium carry out purifying.Ammonium sulfate precipitation and acid or chaotropic agent extract and can be used in the fractional separation of sample.Typical purification step can comprise hydroxyapatite chromatography, size exclusion chromatography, FPLC and RPHPLC (reversed-phase high-performance liquid chromatography).Suitable anionic exchange medium comprises glucan derivative, agarose, Mierocrystalline cellulose, polyacrylamide, property silicon-dioxide, or the like.PEI, DEAE, QAE and Q derivative are preferred, and preferred especially DEAE Fast-Flow Sepharose (Pharmacia, Piscataway, NJ).Typical chromatography media comprises those phenyl, butyl or octyl group deutero-medium, as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia), or the like; Or acrylic resin, as AmberchromCG71 (Toso Haas) etc.Suitable solid support comprises granulated glass sphere, silicone, celluosic resin, sepharose 4B, Sepharose pearl, polystyrene bead, cross-linked polyacrylamide resin or the like, and they are insoluble under the condition that will use.These upholders can be modified with reactive group, make protein partly to be attached on the upholder by amino, carboxyl, sulfydryl, hydroxyl and/or carbohydrate.The example of coupling chemical method comprises cyanogen bromide-activated, N-hydroxy-succinamide activation, epoxide activation, sulfydryl activation, hydrazides activation and is used for the carboxyl and the aminoderivative of carbodiimide coupling chemical method.These and other solid-phase media is well-known and widespread use in the art, and can locate available from product vendor.The method that receptor polypeptides is attached on the supporting dielectric is well-known in the art.The selection of ad hoc approach is the problem of a conventional design, and it partly is by the decision of the characteristic of selected upholder.Consult for example " affinity chromatography: principle and method " (Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988).

Can utilize their characteristic to separate polypeptide of the present invention.For example, immobilized metal absorption (IMAC) chromatography can be used for the protein that purifying is rich in Histidine.Say that simply gel is with divalent-metal ion earlier, form inner complex (E.Sulkowski, biochemical dynamically 3:1-7,1985).The protein that is rich in Histidine will be adsorbed in (this depends on used metal ion) on this matrix with different avidity, and can be by competitive wash-out effect, reduce pH, or with strong chelating agent wash-out.Other purification process comprises with the protein of lectin affinity chromatography and ion exchange chromatography purifying glycosylation (Enzymology method, 182 volumes, " protein purification guide ", M.Deutscher, (volume), 529-539 page or leaf, Acad press, San Diego, 1990).Perhaps, can make up the syzygy of desired polypeptides and affinity labelling (as poly Histidine, maltose binding protein matter, immunoglobulin domains) to help purifying.

Purposes

Polypeptide of the present invention has the structural performance of four-helix bundle cytokine.If a kind of protein is solvable and have that the ability of conducted signal and adjusting cell proliferation is described as cytokine with it usually by the cell surface receptor effect.Cytokine has the folding classification of several tertiary structures, comprises dimer (as Regular Insulin, PDGF), β-trilobal folding (as FGF, IL-1), and the full α four-helix bundle of rich halfcystine.The latter be characterised in that have on the uniqueness-on-down-4 spirals of following topological framework, be labeled as A, B, C and D, wherein two from top to bottom ring connect spiral A and B and spiral C and D.Consult, for example, Manavalan etc., protein chemistry magazine 11 (3): 321-31 (1992).Sometimes the four-helix bundle cytokine further is divided into again short chain (as IL-4, IL-2, GM-CSF) and long-chain (as TPO, tethelin, leptin, IL-10), wherein the latter has long A and D spiral and top-down ring usually.We can use term " cytokine " with the free burial ground for the destitute from now on " and " four-helix bundle cytokine ".The spiral A of Zcyto10 comprises that amino-acid residue 35 (Isoleucine) to amino-acid residue 49 (Isoleucine), also represented by SEQ ID NO:14; Spiral B comprises the 91st amino acids leucine to the 105 amino acids Threonines, is also represented by SEQ ID NO:15; Spiral C comprises the 112nd amino acids residue leucine to the 126 amino acids residue halfcystines, is also represented by SEQ ID NO:16; Spiral D comprises the 158th amino acids residue Xie Ansuan to the 172 amino acids residue methionine(Met)s, is also represented by SEQ ID NO:17.

People Zcyto10 has intramolecular disulfide bond between Cys33 and Cys126.Expect that other 4 halfcystine Cys80, Cys132, Cys81 and Cys134 form two intramolecular disulfide bonds of Cys80-Cys132 and Cys81-Cys134 form.Expectation comprises Cys33, Cys126, Cys80, Cys132, Cys81 and Cys134 to the important residue of the structural stability of Zcyto10.Expecting that any one is mutated into other residue in these residues all can make the Zcyto10 functionally inactive.

The structural stability of Zcyto10 also depends on the hydrophobic surface of keeping on four alpha-helixs that buries.Estimate that residue Ile42, Phe46, Ile49, Leu91, Val94, Phe95, Tyr98, Leu112, Phe116, Ile119, Leu123, Val158, Leu162, Leu165, Leu168, Leu169 and Met172 bury in protein core in the heart, if they change, then the substituted amino acid residue must be a hydrophobic amino acid.

Expection relates to Zcyto10 and cell surface receptor bonded residue and comprises that the Asp57 in the top-down ring and expectation between spiral A and B are exposed to the charged residue Lys160 and the Glu164 on spiral D surface.On protein surface, be the hydrophobic surface piece that contains residue Ile62, Leu71, Ile167 and Trp171 on ring AB and the spiral D zone.These residues may interact with the hydrophobic surface piece on the cell surface receptor.

Have an appointment 28% homogeny of people Zcyto10 polypeptide of the present invention and interleukin-10 (IL-10).Mouse Zcyto10 polypeptide and people IL-10 have about 24% homogeny, with have an appointment 27% homogeny of mouse IL-10.Have an appointment 76% homogeny of people Zcyto10 polypeptide and mouse Zcyto10 polypeptide.

The spiral A of mouse Zcyto10 comprises the 35th amino acids residue Isoleucine to the 49 amino acids residue arginine of SEQ ID NO:19, also represents with SEQ ID NO:21.The spiral B of mouse Zcyto10 comprises the 91st amino acids residue leucine to the 105 amino acids residue Threonines of SEQ ID NO:19, also represents with SEQ ID NO:22.The spiral C of mouse Zcyto10 comprises the 112nd amino acids residue leucine to the 126 amino acids residue halfcystines of SEQ ID NO:19, also represents with SEQ ID NO:23.The spiral D of mouse Zcyto10 comprises the 158th amino acids residue Xie Ansuan to the 172 amino acids residue methionine(Met)s of SEQ ID NO:19, also represents with SEQ ID NO:24.

IL-10 is a kind of cytokine, and it can suppress other production of cytokines, induces activated b lymphopoiesis and differentiation, inhibition HIV-1 to duplicate and IFN-is presented antagonistic effect.As if IL-10 exist with the dimeric forms that is formed by 180 ° of two relevant alpha-helix peptide zones of rotation.Consult, for example, Zdanov etc., structure: 3 (6): 591-601 (1996).Reported that IL-10 is the product of activation Th2 T-cell, B-cell, keratinocyte and monocyte/macrophage, can regulate Th1 T-cell response.Can synthesize by the cytokine that suppresses Th1 T-cell and finish such adjusting.Consult, for example, Hus etc., Int.Immunol.4:563 (1992) and D ' Andrea etc., The Journal of Experimental Medicine (J.Exp.Med.) 178:1042 (1992).It is synthetic to have reported that also IL-10 can suppress the cytokine of natural killer cell and monocyte/macrophage.Consult, for example, Hus etc. (above quoting) and Fiorentino etc., Journal of Immunology, 146:3444 (1991).In addition, found that IL-10 is to the provide protection of insulin-dependent diabetes mellitus tool.

In the tissue distribution of mRNA of new DNA corresponding to this is analyzed, observed single transcript, be approximately 1.2Kb.Utilize Clontech to organize Northern, observing obviously has people's transcript in tracheae, placenta, testis, skin, sialisterium, prostate gland, Tiroidina more, and expresses lower in stomach and pancreas.Zcyto10 is expressed in the following mouse tissue: kidney, skeletal muscle, sialisterium, liver and skin.

The tissue specificity that Zcyto10 expresses shows that Zcyto10 may be growth and/or keep the factor in tracheae and sialisterium, stomach, pancreas and muscle; And may be important in local immune response.And the Zcyto10 assignment of genes gene mapping is on karyomit(e) 1q32.2, and this shows that Zcyto10 is growth/differentiation factor or is important as IL-10 in immune response regulation.

The present invention also provides the reagent that can be used in the diagnostic use.Whether the probe that contains Zcyto 10 DNA or RNA or its subsequence can be used for measuring Zcyto 10 genes and is present on the karyomit(e) 1 or sudden change has not taken place.

The present invention also provides the reagent of the remarkable therapeutic value of tool.Zcyto 10 polypeptide (natural or recombinant forms), its fragment, their antibody and antiidiotypic antibody and be accredited as to sign an undertaking and close the compound of avidity with Zcyto 10 polypeptide, at abnormal physiology or to grow in the treatment of diseases related (comprise abnormality proliferation, change as suffering from cancer or degenerative disease or immunity) be useful.

But anti-Zcyto 10 polypeptide antibodies of purifying also are applied to patient subsequently.For the effect for the treatment of, these reagent can combine with other activity or inert fraction, as stablizer and vehicle harmless on pharmaceutically acceptable carrier or diluent and the physiology.These mixtures can be through sterile filtration, and place reagent bottle or be stored in stable water preparation by lyophilize and place with dosage form.The invention still further relates to antibody and binding fragment thereof or comprise the purposes of the single-chain antibody of non-complement combining form.

Effectively treatment must reagent deal depend on many different factors, comprise application method, target site, patient's physiology state and the other medicines of using.Therefore, therapeutic dose should be determined so that security and validity reach best.In general, the external using dosage of these reagent can provide the guidance of usefulness for the significant quantity of using in its body.The animal experiment for the treatment of the effective dose of the special imbalance used dosage of will behaving provides further emissary indication.That insecticide-applying way comprises is oral, intravenously, intraperitoneal, muscle, use into lung or tracheae with Sprayable through the skin medication or by atomizer or spraying gun.Pharmaceutically acceptable carrier will comprise water, salt solution, some damping fluids etc.Usually the dosage range of expection is a 1-1000 μ g/Kg body weight/day.Yet dosage can be higher or lower, and this can be determined by the physician of this area routine techniques.The complete discussion of formula of medicine and dosage range can be consulted Remington ' s PharmaceuticalScience, the 18th edition (Mack Publishing Co., Easton, Penn., 1996), and Goodman and Gilman: the pharmacology basis of treatment, the 9th edition (Pergamon Press1996).

Treatment based on nucleic acid

If mammiferous Zcyto 10 transgenations or disappearance can be introduced this mammiferous cell with Zcyto 10 genes.In one embodiment, the gene of coding Zcyto 10 polypeptide is introduced in the body on a virus vector.Such carrier comprises attenuation or defective type dna virus, such as (but being not limited to) simple sore exanthema virus (HSV), papilloma virus, epstein-Barr virus (EBV), adenovirus, adeno associated virus (AAV) or the like.The preferred defective virus that lacks virogene fully or almost completely.Defective virus does not have infectious after introducing cell.Use the defective virus carrier to give and cell, and do not worry that this carrier can infect other cell at specific regional area.Special carrier example comprises, but be not limited to, the adenovirus carrier of defective herpesvirus 1 (HSV1) carrier [Kaplitt etc., Molec.Cell.Neurosci., 2:320-330 (1991)], attenuation, as by Stratford-Perricaudet etc., Journal of Clinical Investigation (J.Clin.Invest.), the described carrier of 90:626-630 (1992), and defective type adeno-associated virus vector [Samulski etc., Journal of Virology, 61:3096-3101 (1987); Samulski etc., Journal of Virology, 63:3822-3828 (1989)].

In another embodiment, gene can be introduced in retroviral vector, as described in following document: No. the 5th, 399,346, the United States Patent (USP) of Anderson etc.; Mann etc., cell, 33:153 (1983); No. the 4th, 650,764, the United States Patent (USP) of Temin etc.; No. the 4th, 980,289, the United States Patent (USP) of Temin etc.; Markowitz etc., Journal of Virology, 62:1120 (1988); No. the 5th, 124,263, the United States Patent (USP) of Temin etc.; International Patent Publication No. WO 95/07358, open by people such as Dougherty March 16 nineteen ninety-five; Hematology, 82:845 (1993).Perhaps, carrier can utilize liposome to pass through in the body lipofection to introduce.The synthetic cation lipid can be used to prepare liposome with transfection in the body that is used for the label coding gene [Felgner etc., institute of NAS newspaper, 84:7413-7417 (1987); Consult Mackey etc., institute of NAS newspaper, 85:8027-8031 (1988)].Use lipofection foreign gene to be introduced the special organ has some practical benefit in the body.Liposome has been represented the one side of advantage to the molecular targeted property of specific cells.Obviously, instruct another aspect from advantage to the transfection of special cells that represented.Also be clear that very much, at the heterogeneous tissue of cell, in pancreas, liver, kidney and brain, especially favourable to the directed transfection of special cells type.For the purpose of target can be with other molecule in the lipid chemical coupling.The target peptide, as hormone or neurotransmitter, and as the protein of antibody and so on, or non-peptide molecule all can with the liposome chemical coupling.These liposomes also can be applied to lung or tracheae with Sprayable with the method for atomizer or spraying gun.

Cell is taken out in body, will introduce in this cell as the carrier of naked DNA plasmid then, and subsequently this transformant be implanted again, such operation is possible.The naked DNA carrier introducing purpose host cell that can will be used for gene therapy by technology known in the art, [consult as the use of transfection, electroporation, microinjection, transduction, cytogamy, deae dextran, calcium phosphate precipitation, particle gun or the use of dna vector translocator, as Wu etc., journal of biological chemistry 267:963-967 (1992); Wu etc., journal of biological chemistry, 263:14621-14624 (1988)].

But Zcyto 10 polypeptide also can be used for preparing the antibody of specific combination Zcyto 10 polypeptide.These antibody can be used for preparing antiidiotypic antibody subsequently." antibody " used herein speech comprises polyclonal antibody, monoclonal antibody and such as F (ab) ₂With the Fab of Fab fragment and so on, or the like, comprising genetic engineering antibody.If antibody and Zcyto 10 polypeptide bonded Ka 〉=10 ⁷/ M, but then this antibody is defined as specific combination.This area routine techniques personnel can measure the avidity (consulting, for example, Scatchard, the same) of monoclonal antibody easily.

Preparation polyclone and monoclonal antibody method are well known in the art (for example consults Sambrook etc., molecular cloning: laboratory manual (second edition) (cold spring port, New York, 1989); And Hurrell, J.G.R., editor, the mono-clonal hybridoma antibody: technology and application (CRC Press, Inc., Boca Raton, FL, 1982) all are hereby incorporated by).This area the routine techniques personnel can obviously see, polyclonal antibody can be from various warm-blooded animals, as preparing in horse, ox, goat, sheep, dog, chicken, rabbit, mouse and the rat.By use such as Fu Shi fully or the adjuvant the Freund can improve the immunogenicity of Zcyto 10 polypeptide.The known various tests of those skilled in the art all can be used for detecting can with the antibody of Zcyto 10 polypeptide specific combination.The exemplary experimental details is in " antibody: laboratory manual ", Harlow and Lane (volume) (press of cold spring harbor laboratory, 1988).The representative example of these tests comprises: merge the test of immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), Dot blot test, inhibition or competition experiments and sandwich.

Anti-Zcyto 10 antibody can be used for this proteinic cell of marker expression, are used for affinity purification, are used to measure the cyclical level of soluble protein polypeptide in diagnositc analysis, and can be used as antagonist to block external and intravital part combination and signal transduction.

Another aspect of the present invention provides and has contained purifying Zcyto 10 polypeptide and medicinally accept carrier-bound pharmaceutical composition.To further discuss as this paper, these compositions are used in prevention and treat and regulate cell proliferation, cytodifferentiation or cytokine generation in the disease that it is characterized by improper cell proliferation, cytodifferentiation or cytokine generation.In addition, Zcyto 10 polypeptide of the present invention can be used in tracheae is special or tracheal bronchus the is special application, as tracheal bronchus epithelium or its basal cell keep or wound repair, adjusting mucus produces or the mucus of residue is removed or other disease in treatment asthma, bronchitis or tracheal bronchus road.The application dosage scope of estimating Zcyto 10 polypeptide with the used identical dosage of Zcyto 10-FC construct to than it between high 100 multiple doses, this depends on the stability of Zcyto 10 polypeptide.The therapeutic dose of Zcyto 10 is 5-5000 μ g/Kg/ days.

Zcyto 10 polypeptide high expression levels of the present invention are found to be present in the saliva by the Western engram analysis in sialisterium and tracheae.The numerous protein that sialisterium is synthetic and secretion has the different biological function.Such protein helps lubricated (as Saliva Orthana and rich proline protein matter), the Remineralization (as statherin and ionic rich proline protein matter) in oral cavity and digests (as amylase, lipase and proteolytic enzyme) and confession under directions antimicrobial (as rich proline protein matter, N,O-Diacetylmuramidase, histatin and lactoperoxidase) and mucous membrane composition to keep (as Saliva Orthana) ability.In addition, saliva is the abundant source of the sialisterium somatomedin of synthesizing.For example, known saliva contains Urogastron (EGF), nerve growth factor (NGF), transforminggrowthfactor-(TGF-α), transforming growth factor-beta (TGF-β), Regular Insulin, insulin-like growth factor I and II (IGF-I and IGF-II) and fibroblast growth factor (FGF).Consult, for example, Zelles etc., J.Dental.Res.74 (12): 1826-32,1995.The process of the sialisterium synthetically grown factor is considered to male sex hormone dependent form, is essential for oral cavity and GI health.

Thereby Zcyto 10 polypeptide, its stimulant or antagonist have treatment effectiveness in gi tract or oral cavity regeneration.In order to confirm that Zcyto polypeptide of the present invention, stimulant or antagonist have such ability, assess the ability of these Zcyto 10 polypeptide, stimulant or antagonist degraded starch by means known in the art.By intervening the adjusting that the lymphocytic intersection of Th1 and Th2 is regulated the growth, differentiation and the cytokine generation that reach other inflammatory cellular mediators such as eosinophil, mastocyte, basophilic cell, neutrophilic granulocyte and scavenger cell, Zcyto 10 polypeptide, its stimulant or antagonist can be used for treating asthma and other tracheal bronchus tract disease.The tonus of muscle in Zcyto 10 polypeptide and stimulant thereof or the also adjustable solar term pipe of the antagonist bronchial.

In the time of in mixing ointment or frost, Zcyto 10 polypeptide also can be used for treating many systematicness or partial tetter, for example are generally eczema, psoriasis or xeroderma, or the skin care that is used to be correlated with.Zcyto 10 polypeptide also can be injected directly into the myatrophy of muscle with treatment old man, patient or long-term bed person in addition.

It is the somatocyte genetic technique [Cox etc., science 250:245-250 (1990)] that is used to make up the chromosomal high resolving power precession diagram of Mammals that radiation hybridization is drawn.Know that partially or completely gene order just can design the PCR primer that is suitable for the use of karyomit(e) radiation hybridization plotting sheet.Cover the radiation hybridization plotting sheet of whole people's gene group, (HuntsVille AL) can buy and obtains for Research Genetics, Inc. as Stanford G3 RH Panel and GeneBridge 4 RH Panel.But these plate PCR-based and carry out chromosomal localization and gene, sequencetagged site (STS) and other non-polymorphism or the arrangement of polymorphism sign in the purpose zone apace.This comprises the proportional physical distance of directly setting up between new discovery goal gene and original icon sign.Know that gene location all is useful in many aspects, comprise: determine 1) whether one section sequence is the part of an existing contig, and obtain genetic sequence around other of such as YAC-, BAC-or cDNA clone various ways, 2) provide possible candidate gene for the chain genetic diseases of demonstration and identical chromosomal region, and 3) cross reference model biology, such as mouse, with the function of assisting to determine that special gene is had.

The result shows Zcyto 10 genetic mappings 889.26cR-3000 from human chromosome 1 linkage group top on WICGR radiation hybridization collection of illustrative plates.Nearside or distally framework mark are respectively DIS504 and WI-9641 (DIS2427).Mark is with lq32.2 zone (hereditary location database, University of Southampton, the www server: http://cedar.genetics.soton.ac.uk/public-html/) of Zcyto 10 assignments of genes gene mapping on complete LDB karyomit(e) 1 collection of illustrative plates around utilizing.Many genes have been drawn in the 1q32.2 zone of karyomit(e) 1.Especially, the sudden change in this zone has found to cause van der Woude syndromes, and is relevant with relevant with cleft palate sometimes lower lip deformity.Thereby, be expressed in the gene therapy that Zcyto 10 genes in the sialisterium can be used for this syndromes.If Mammals Zcyto 10 transgenations or disappearance can be introduced the Zcyto10 gene in this mammiferous cell.

Another aspect of the present invention relates to the NO:1 with SEQ ID, the section complementary antisense polynucleotides composition of 3,18 and 33 listed polynucleotide.Can design such synthesising antisense scant nucleotide with in conjunction with the mRNA of coding Zcyto 10 polypeptide and suppress the translation of this mRNA.Such antisense oligonucleotide can be used for suppressing Zcyto 10 peptide coding expression of gene among cell culture or the subject.

The present invention also provides useful reagent in diagnostic use.For example, whether Zcyto 10 genes, the probe that contains Zcyto 10 DNA or RNA or its subsequence can be used for detecting Zcyto 10 genes and are present on the karyomit(e) 1 or sudden change has not taken place.Detected chromosome aberration on Zcyto 10 gene locations changes and rearrangement including, but not limited to aneuploid, gene copy number change, insertion, deletion, restriction site.By molecular genetic technique, the STR (STR) of analyzing, use round pcr such as restriction fragment length polymorphism (RFLP) analyzes and other genetic linkage analysis technology known in the art [Sambrook etc., the same; Ausubel etc., the same; Marian, A.J., Chest, 108:255-265, (1995)], can detect such distortion with polynucleotide of the present invention.

Those persons skilled in the art of this area will recognize that the sequence that is disclosed among the SEQ ID NO:2,4,12,13,19,20,25,26,34 and 35 represented the single allelotrope of people and mouse Zcyto 10 genes and polypeptide, and recognize and can have allelic variation and other montage mode.By standard step by cloning allelic variant to surveying from the cDNA of Different Individual or genomic library.The allelic variant of dna sequence dna shown in the SEQ ID NO:1,3,18 and 33 comprises variant that contains silent mutation and the variant that suddenlys change and cause aminoacid sequence to change, all within the scope of the present invention.

Zcyto 10 sequences non-ly turn over that position 706,813,855 and 906 places have 7 courier's unstable primitives in the pool district at 3 ' of SEQ ID NO:1.Handle the cell of expressing Zcyto10 with cycloheximide and can weaken this courier's unstable.Consult Shaw, G. etc., cell 46:659-667 (1986).In addition, can on gene, change or remove the 3 ' non-translational region of rich AT with further raising courier stability.

Use Zcyto 10 to promote wound healing

The data presentation Zcyto 10 of embodiment 4 works in wound healing.Therefore, Zcyto10 can be applicable to promote wound healing in wound or the burn.Zcyto 10 can 1-100 μ g/Kg body weight sosimetric system use.Zcyto 10 also can be applied on the wound by ointment or the ointment mode that contains 1ng-1mg Zcyto 10/g ointment or ointment.Consult Remington ' sPharmaceutical Sciences, the 18th edition (Mark Publishing Co., Easton, Penn., 1996).Should every day Zcyto 10 be used on the clean wound up to wound healing.

Utilize Zcyto 10 platelet increasing numbers

As visible in following examples 7, we have found that Zcyto 10 can be used for the platelet increasing number.This is for because chemotherapy or radiotherapy and thrombocytopenic cancer patient is even more important.Zcyto 10 can use in treatment with the medicinal carrier of accepting.

The present invention further is described by following unrestricted embodiment.

Embodiment 1

The clone of Zcyto 10

The full length sequence of Zcyto10 * 1 (more microscler formula) and Zcyto10 * 2 (than short-form) is illustrated by following steps: carry out 3 ' RACE , and two sequencing fragments that will produce (SEQ ID NO:10 and SEQ ID NO:11), then with computer with the est sequence shown in the SEQ ID NO:5 with manually be stitched together from the segmental overlap of two 3 ' race.

Oligonucleotide zc15907 (SEQ ID NO:6) is designed to just be positioned at Zcyto 10 infers methionine(Met) upstream region (5 ').Another oligonucleotide zc15906 (SEQ IN NO:7) design in farther downstream, just is positioned at the upstream region of signal sequence cleavage site.These oligonucleotide are used for 3 ' the RACE reaction of people's tracheae marathon cDNA.ZC15907 is used for the 1 ' the race reaction, zc15906 is used for nested type 3 ' race reaction.Begin with the people's tracheae mRNA available from Clontech, (Clontech, Palo Alto CA) prepare MARATHON cDNA by manufacturer specification with Marathon cDNA amplification kit.

Specification sheets by manufacturers in the Marathon cDNA amplification kit carries out the PCR reaction, and some modifications are arranged on thermal circulation parameters.The loop parameter that is used for PCR reaction is:

94 ℃ 1 minute 30 seconds, 1 time

94 ℃ 15 seconds, 68 ℃ 1 minute, 30 times

72 ℃ 7 minutes, 1 time

The loop parameter that is used for nested PCR reaction is: 94 ℃ 1 minute 30 seconds 1 time, 94 ℃ 15 seconds 68 ℃ 1 minute 20 seconds, 30 times, 72 ℃ 7 minutes 1 time.

The product that produces is carried out 1.2% sepharose (Gibco agarose) electrophoresis, and visible two master tapes are approximately at a distance of 80bp.These bands are cut out and by manufacturer specification QIAEX ^TMResin (Qiagen) carries out gel-purified.With these sequencing fragments, can discern the full length sequence of Zcyto 10 then.

Embodiment 2

Rna blot analysis

Organize trace I, II, III and RNA principal point trace (Master DotBlot) to the people (Clontech) are surveyed more, to determine the tissue distribution of Zcyto 10.Design 45-mer antisense oligonucleotide (SEQ IDNO:9) and be used for detection with est sequence (SEQ ID NO:5bp 100-145).

SEQ ID NO:9 with 15pm carries out with T4 polynucleotide kinase (Gibco-BRL) ³²The P end mark.Labeled reactant liquid contains 2 μ l 5X kinase reaction damping fluids (Gibco-BRL), 1 μ l T4 kinases, 15pm SEQ ID NO:9,1 μ L 6000Ci/mmol ³²P γ-ATP (Amersham) and water to 10 μ l.37 ℃ of incubations of reaction solution 30 minutes.Remove uncorporated radioactivity with NucTrapProbe Purification Column (Stratagene).Organize RNA trace and people RNA master trace (Clontech) 50 ℃ of prehybridizations 3 hours in 10ml ExpressHyb more, wherein contain 1mg salmon sperm DNA and 0.3mg people cotl DNA (Gibco-BRL), the two all boiled 3 minutes, and ice is put and added among the ExpressHyb then in 2 minutes.Spend the night 50 ℃ of hybridization.Initial wash conditions is as follows: 2 * SSC, 0.1%SDS room temperature 40 minutes, wherein repeatedly change washings, and 1 * SSC, 0.1%SDS were 64 ℃ (Tm-10) 30 minutes then.Trace is subsequently to exposure two days.

The expression of Zcyto 10 is presented at the band that 1.2kb is arranged in the tracheae approximately in the RNA trace, and weak 1.5kb band is arranged under one's belt, and the more weak band of these two kinds of sizes is arranged in pancreas.Dot blot is presented at and has Zcyto 10 in tracheae, sialisterium, placenta, testis, skin, prostate gland, suprarenal gland and the Tiroidina.

In mouse, Zcyto 10 finds to be present in kidney, skeletal muscle, sialisterium, liver and the skin.

Embodiment 3

The karyomit(e) of Zcyto 10 distributes and the location

(Hantsville AL) draws Zcyto 10 on No. 1 karyomit(e) for Reseach Genetics, Inc. with " the Stanford G3 radiation hybridization plotting sheet " that can buy.Should " Stanford G3 RH plate " but comprise from each PCR DNA among 83 radiation hybridization clones of whole people's gene group, add two contrast DNA (RM donor and A3 acceptor).A spendable www server of the public (http://shgc-www.stanford.edu) can be used for the chromosomal localization of mark.

In order to draw to Zcyto10 with " Stanford G3 RH plate ", but 96 hole titer plate (Stratagene at PCR, La Jolla sets up the reaction system of 20 μ l on CA), uses it on " RoboCycler Gradient96 " thermal cycler (Stratagene).During 85 PCR react each is composed as follows: 2 μ l 10x KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., Palo Alto, CA), 1.6 μ l dNTP mixed solution (every kind of 2.5mM, PERKIN-ELMER, Foster City, CA), 1 μ l has adopted primer (SEO ID NO:6,5 ' ATT CCT AGC TCC TGT GGT CTC CAG 3 '), 1 μ l antisense primer (SEQ ID No:8,5 ' TCC CAA ATT GAG TGT CTT CAG T3 '), 2 μ l " Redi Load " (Research Genetics, Inc.,-Huntsville, AL), 0.4 μ l 50x Advantage KlenTaq Polymerase Mix (ClontechLaborabories, Inc.), 25ng DNA from single hybridization clone or contrast uses ddH ₂O is supplemented to cumulative volume 20 μ l.Cover and sealed reaction liquid with equivalent mineral oil.The PCR cycling condition is as follows: first circulation of beginning is 95 ℃ of sex change 5 minutes, then 35 circulations be 95 ℃ of sex change 1 minute, 66 ℃ of annealing 1 minute again 72 ℃ extended 1 minute, last circulation is 72 ℃ and extended 7 minutes.(Life Technologies, Gaithersburg MD) go up the electrophoresis reaction product isolated at 2% sepharose.

The result shows that Zcyto 10 and framework mark SHGC-36215 are chain, and tool is greater than 10 LOD value, and range mark is 14.67CR-10000.By means of mark on every side, Zcyto10 is positioned 1q32.2 zone (the The GeneticLocation Database of No. 1 map of complete LDB, University of Southampton, www server: http://cedar.genetics.soton.ac.uk/public_html/).

Embodiment 4

Utilize Zcyto 10 to promote wound healing

Used the female Balb/C mouse of normal mature in this research.They are placed the animal care chamber with the circulation of 12 hours light-dark, feedwater and feed during studying in the random drinking water mode of laboratory rodents.They were closed the cage respectively from operating that day.

On surgical operation same day, be used in 104mg/kg ketamine (Vetalar, Aveco Inc. in aseptic (0.2 μ-filtration) phosphate buffered saline(PBS) (PBS), Ft.Dodge IA) adds 7mg/kg xylazine (Rompun, Mobey Corp., Shawnee is KS) by the peritoneal injection anesthetized animal.Also (Carter-Wallace, NewYork NY) make skin depilatory, clean with water then with NAIR  to cut the back hair.100% aloe vera gel is used to resist because the alkaline burn due to the NAIR  processing places animal then on the recirculated water warm table and reaches fur drying on every side up to skin.

Use metofane (Pittman Moore, Mundelein, NJ) anesthetized animal and the back of losing hair or feathers then with 70% alcohol wipe.Skin and sarcolemma above chest lumbar vertebrae level is passed the other zone of backbone are made 4 otch, each 0.5 square centimeter.BIOCLUSIVE  (the Johnson ﹠amp of section is applied in semipermeability sealing with viscosity; Johnson, Arlington TX) covers the wound and the skin that loses hair or feathers on every side.In order to assess closed parameter after a while, notching edge is retouched on the acetic acid transparent paper by BIOCLUSIVE .

Handle contrast skin and compromised skin with Qiagen RNeasy Midi Kit in different time points (7 hours, 15 hours and 24 hours).Speak briefly, skin (contrast and injured area) is weighed and homogenate in appropriate volume lysis buffer (RLT).The rotation lysate is organized residue with removal and equal-volume 70% ethanol is added; Upper prop behind the mixing.Sample rotation 5 minutes is also washed 1 time with the 3.8mlRW1 damping fluid, washes (each 2.5ml) twice with RPE then.With the total RNA of no RNase water elution.Expression level with PCR in real time (Perkin Elmer ABI Prisn 7700 SequenceDetector) detection of skin sample.

Experimental design is a no template contrast, a cover standard and a skin samples.Total RNA makes typical curve with the mouse kidney.The three cover total RNA of skin (25ng) are used for this experiment: 7 hours (contrast and injured); 15 hours (contrast and injured), 24 hours (contrast and injured).Each sample is undertaken by a step reverse transcription PCR triplicate on 7700 sequential detection instrument.Built-in (in-house) forward primer SEQ ID NO:36, reverse primer SEQ IDNO:37 and Perkin Elmer ' s Taq Man probe (ZG-7-FAM) have been used in this experiment.The condition of one step reverse transcription PCR is as follows: (reverse transcription step) 48 ℃ 30 minutes, (40 round-robin PCR steps) 95 ℃ 10 minutes, 95 ℃ 15 seconds, 60 ℃ 1 minute.

In the contrast skin samples when 7 hours and 15 hours the expression level of Zcyto10 similar, be respectively 2.46ng/ml and 2.61ng/ml.The expression level of Zcyto10 is 0 in 24 hours the contrast skin samples.The Zcyto10 expression level of compromised skin is 5.17ng/ml (having improved more than 2 times with comparing of control sample) in the time of 7 hours.Zcyto 10 expression levels of compromised skin are 14.45ng/ml (compare increased by 5.5 times with control samples) in the time of 15 hours.Zcyto 10 expression levels of compromised skin are 5.89ng/ml in the time of 24 hours.Repeated experiments also comprises negative control (yeast tRNA), shows similar tendency, and the result of yeast tRNA is near 0.The result shows that amplification exists really and is the mouse specificity.

These data show that Zcyto10 works in wound repair, because the Zcyto10 expression level of wounded tissue increases in time and reduces than the high of control sample and its.Therefore, Zcyto10 can be applicable to injury to promote wound healing.

Embodiment 5

Transgenic mice

The transgenic mice of Zcyto10 is expressed in production under white protein or metallothionein promoter control.During birth, several mouse have shinny outward appearance, and it is limited to move.The skin of these mouse is tight and wrinkle is arranged, and wherein several also have filament class hair on lower lip.Some is swollen for nostril and mouth zone, four limbs and afterbody.

Use a transgenic mice of white protein promotor to survive 3 days, it is delayed growth seriously.No ear is grown, and the growth of toes delays.When it is dying at the 1st, 2 or 3 day, put to death all animals.Collect tail and liver sample and with its anchored in place in 10% neutral formalin and be embedded in the paraffin, be cut into the section of 3 μ m and use H﹠amp; E dyeing.All mouse of this phenotype of tool are transgenic mice and have Zcyto10 from low to high to express.

In removing extracutaneous most tissues, do not observe significant change.It is thick that the skin of those mouse of the high Zcyto10 expression level of young mouse, especially tool of expression Zcyto10 tends to do not have the skin of expressing young mouse.The granular layer of these young mouse is not compared the thickness minimizing with there being the young mouse of expression, and spinous layer is thicker because cellular layer increases and/or cell dia increases.

The change in epidermis, the corium of the mouse that the medium Zcyto10 of tool expresses is by the appropriateness expansion of viscous substance focus ground.

Embodiment 6

From cell culture medium purifying Zcyto10

With cation-exchange chromatography and the size exclusion chromatography two-stage process Zcyto10 that separation of C HO cell produces in cell culture medium.

A. cation-exchange chromatography step

Material therefor

A kind of TOYOPEARL ion exchange resin of apparatus covalent bonding sulfopropyl (SP), 2.2cm diameter (D) * 6cm height (H) post (AMICON) that the SP-650M Zeo-karb is filled.

Collect to control oneself 15 liters of substratum of young hamster kidney (BHK) cell that contained the Zcyto plasmid transfection.With 2N HCl medium pH is transferred to pH5.With 50mM sodium acetate (NaAc, pH5.0) the above-mentioned packed column of balance.With about 8ml/ minute 20 column volume (CV)/hour speed with the substratum pillar of packing into.With the 50mM NaAc of 10 column volumes, pH5.0 washed post after upper prop was finished.Use 50mM NaAc then, the material in the 20 column volume NaCl gradient elution posts among the pH5.0.The NaCl gradient is 0 → 0.5M NaCl.This is concentrated into 170ml with the material in the substratum from 15 liters.

(Millipore, Inc.Bedford MA) further are concentrated into about 5ml with the 170ml cutting that produces with 5000 the rotating centrifugal thickener of damming.

B. size exclusion (S-100) gel-filtration step

Material therefor

(1.6cm diameter) * 93cm (height) post

The S-100 gel (Pharmacia, Piscataway, NJ)

Subsequently 5ml is gathered in the crops on the pillar that liquid is added to the above-mentioned S-100 of containing gel.This pillar is about 7.0 with 5X phosphate buffered saline(PBS) balance to post pH.With 1 * PBS with 1.5ml/ minute flow velocity with Zcyto10 and separated from contaminants.Collect each component with the 2ml amount.By determining that with blue painted sodium lauryl sulphate (SDS) polyacrylamide gel electrophoresis of coomassie the Zcyto10 polypeptide washes among the component 52-64 when wash-out begins about 90 minutes.There is a band at the expection molecular weight place that gel is presented at about 14KDa.

Embodiment 7

The clone of mouse Zcyto10

5 ' MARATHON RACE ^TM(PCR primer CA) is for being attached to MARATHON for Clontech, Palo Alto ^TMThe SEQ ID NO:38 of AP1 linker, it be attached to AP2MARATHON ^TMThe SEQ ID NO:39 of linker is nested, 3 ' MARATHON RACE ^TMPrimer for being attached to MARATHON RACE ^TMThe SEQ ID NO:40 of AP1 linker, it be attached to MARATHON RACE ^TMThe SEQ ID NO:41 of AP2 linker is nested, to mouse skin MARATHON RACE ^TMCDNA carries out 5 ' and 3 ' PCR.To carry out gel-purified and order-checking from several fragments of these reactions, can illustrate complete encoding sequence and some 5 ' and 3 ' the UTR sequences of mouse Zcyto10.Find two mouse Zcyto10 variants, be called SEQ ID NO:18 and 19 and SEQ ID NO:33 and 34.Thing primer SEQ ID NO:42 and these clones of 43 pcr amplifications.

Embodiment 8

By adenovirus Zcyto10 is applied to normal mouse

Use Zcyto10 by the adenovirus that contains the Zcyto10 gene.As described below have three groups of mouse.Adenovirus is gone into the male and female mice of C57B1/6 from intravenous injection.The tap water that contains bromodeoxyribouridine (Brdu) in execution all mouse acceptance in preceding 3 days.Can detect cell proliferation by Histological method like this.Detect parameters comprises body weight change, complete blood count, serum chemistry, histology, organ weight and the cell proliferation that detects by BrdU.

Experimental design

First group of Zcyto 10 * 1 (SEQ ID NO:18)/pAC-CMV/AdV

1 * 10 ¹¹Particle/agent

(putting to death 9 female, 9 male mices at the 21st day)

(putting to death 2 female, 2 male mices at the 11st day)

Sum=22 mouse

Second group of empty CMV/AdV contrast

1 * 10 ¹¹Grain/agent

(putting to death 10 female, 10 male mices at the 21st day)

(putting to death 2 female, 2 male mices at the 11st day)

Sum=24 mouse

The 3rd group not treated

(5 female, 5 male mices)

Sum=10

The result

The most noticeable result is, compares with empty adenovirus contrast, and observing platelet count in the male and female mice of handling with the Zcyto10-adenovirus significantly increases.This is accompanied by hematocrit reduction and spleen and liver in male mice heavily increases.Thymic weight also reduces in male.The formation contrast is therewith, compares with empty virus control, and the female mice that the Zcyto10 adenovirus is handled shows that white blood cell count(WBC) significantly increases, and mainly is the increase of lymphocyte and neutrophil leucocyte counting.

These results show that Zcyto10 handles influences hemopoietic, but increases this effect both sexes all have except platelet count, and other influence is a sex-specific.

Other influence comprises following result:

Glucose level female in processed group reduces, and male glucose level shows no noticeable change.

Processed group male and female in blood urine nitrogen (BUN) all improve.

Alkaline phosphatase female in processed group is higher, and male demonstration does not have noticeable change.

Processed group male and female middle platelet count is all higher.

Total leukocyte counting (WBC) higher and male demonstration female in processed group does not have noticeable change.

Sequence table

<110>ZymoGenetics，Inc.

＜120〉mammalian sytokine-like polypeptide 10

<130>97-72PC

<160>43

<170>FastSEQ?for?Window?Version?3.0

<210>1

<211>926

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(45)...(572)

<400>1

ctttgaattc?ctagctcctgtggtctccag?atttcaggcc?taag?atg?aaa?gcc?tct 56

Met?Lys?Ala?Ser

1

agt?ctt?gcc?ttc?agc?ctt?ctc?tct?gct?gcg?ttt?tat?ctc?cta?tgg?act 104

Ser?Leu?Ala?Phe?Ser?Leu?Leu?Ser?Ala?Ala?Phe?Tyr?Leu?Leu?Trp?Thr

5 10 15 20

cct?tcc?act?gga?ctg?aag?aca?ctc?aat?ttg?gga?agc?tgt?gtg?atc?gcc 152

Pro?Ser?Thr?Gly?Leu?Lys?Thr?Leu?Asn?Leu?Gly?Ser?Cys?Val?Ile?Ala

25 30 35

aca?aac?ctt?cag?gaa?ata?cga?aat?gga?ttt?tct?gac?ata?cgg?ggc?agt 200

Thr?Asn?Leu?Gln?Glu?Ile?Arg?Asn?Gly?Phe?Ser?Asp?Ile?Arg?Gly?Ser

40 45 50

gtg?caa?gcc?aaa?gat?gga?aac?att?gac?atc?aga?atc?tta?agg?agg?act 248

Val?Gln?Ala?Lys?Asp?Gly?Asn?Ile?Asp?Ile?Arg?Ile?Leu?Arg?Arg?Thr

55 60 65

gag?tct?ttg?caa?gac?aca?aag?cct?gcg?aat?cga?tgc?tgc?ctc?ctg?cgc 296

Glu?Ser?Leu?Gln?Asp?Thr?Lys?Pro?Ala?Asn?Arg?Cys?Cys?Leu?Leu?Arg

70 75 80

cat?ttg?cta?aga?ctc?tat?ctg?gac?agg?gta?ttt?aaa?aac?tac?cag?acc 344

His?Leu?Leu?Arg?Leu?Tyr?Leu?Asp?Arg?Val?Phe?Lys?Asn?Tyr?Gln?Thr

85 90 95 100

cct?gac?cat?tat?act?ctc?cgg?aag?atc?agc?agc?ctc?gcc?aat?tcc?ttt 392

Pro?Asp?His?Tyr?Thr?Leu?Arg?Lys?Ile?Ser?Ser?Leu?Ala?Asn?Ser?Phe

105 110 115

ctt?acc?atc?aag?aag?gac?ctc?cgg?ctc?tgt?cat?gcc?cac?atg?aca?tgc 440

Leu?Thr?Ile?Lys?Lys?Asp?Leu?Arg?Leu?Cys?His?Ala?His?Met?Thr?Cys

120 125 130

cat?tgt?ggg?gag?gaa?gca?atg?aag?aaa?tac?agc?cag?att?ctg?agt?cac 488

His?Cys?Gly?Glu?Glu?Ala?Met?Lys?Lys?Tyr?Ser?Gln?Ile?Leu?Ser?His

135 140 145

ttt?gaa?aag?ctg?gaa?cct?cag?gca?gca?gtt?gtg?aag?gct?ttg?ggg?gaa 536

Phe?Glu?Lys?Leu?Glu?Pro?Gln?Ala?Ala?Val?Val?Lys?Ala?Leu?Gly?Glu

150 155 160

cta?gac?att?ctt?ctg?caa?tgg?atg?gag?gag?aca?gaa?taggaggaaa 582

Leu?Asp?Ile?Leu?Leu?Gln?Trp?Met?Glu?Glu?Thr?Glu

165 170 175

gtgatgctgc?tgctaagaat?attcgaggtc?aagagctcca?gtcttcaata?cctgcagagg 642

aggcatgacc?ccaaaccacc?atctctttac?tgtactagtc?ttgtgctggt?cacagtgtat 702

cttatttatg?cattacttgc?ttccttgcat?gattgtcttt?atgcatcccc?aatcttaatt 762

gagaccatac?ttgtataaga?tttttgtaat?atctttctgc?tattggatat?atttattagt 822

taatatattt?atttattttt?tgctattaat?gtatttaatt?ttttacttgg?gcatgaaact 882

ttaaaaaaaa?ttcacaagat?tatatttata?acctgactag?agca 926

<210>2

<211>176

<212>PRT

＜213〉people

<400>2

Met?Lys?Ala?Ser?Ser?Leu?Ala?Phe?Ser?Leu?Leu?Ser?Ala?Ala?Phe?Tyr

1 5 10 15

Leu?Leu?Trp?Thr?Pro?Ser?Thr?Gly?Leu?Lys?Thr?Leu?Asn?Leu?Gly?Ser

20 25 30

Cys?Val?Ile?Ala?Thr?Asn?Leu?Gln?Glu?Ile?Arg?Asn?Gly?Phe?Ser?Asp

35 40 45

Ile?Arg?Gly?Ser?Val?Gln?Ala?Lys?Asp?Gly?Asn?Ile?Asp?Ile?Arg?Ile

50 55 60

Leu?Arg?Arg?Thr?Glu?Ser?Leu?Gln?Asp?Thr?Lys?Pro?Ala?Asn?Arg?Cys

65 70 75 80

Cys?Leu?Leu?Arg?His?Leu?Leu?Arg?Leu?Tyr?Leu?Asp?Arg?Val?Phe?Lys

85 90 95

Asn?Tyr?Gln?Thr?Pro?Asp?His?Tyr?Thr?Leu?Arg?Lys?Ile?Ser?Ser?Leu

100 105 110

Ala?Asn?Ser?Phe?Leu?Thr?Ile?Lys?Lys?Asp?Leu?Arg?Leu?Cys?His?Ala

115 120 125

His?Met?Thr?Cys?His?Cys?Gly?Glu?Glu?Ala?Met?Lys?Lys?Tyr?Ser?Gln

130 125 140

Ile?Leu?Ser?His?Phe?Glu?Lys?Leu?Glu?Pro?Gln?Ala?Ala?Val?Val?Lys

145 150 155 160

Ala?Leu?Gly?Glu?Leu?Asp?Ile?Leu?Leu?Gln?Trp?Met?Glu?Glu?Thr?Glu

165 170 175

<210>3

<211>793

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(45)...(497)

<400>3

ctttgaattc?ctagctcctg?tggtctccag?atttcaggcc?taag?atg?aaa?gcc?tct 56

Met?Lys?Ala?Ser

1

agt?ctt?gcc?ttc?agc?ctt?ctc?tct?gct?gcg?ttt?tat?ctc?cta?tgg?act 104

Ser?Leu?Ala?Phe?Ser?Leu?Leu?Ser?Ala?Ala?Phe?Tyr?Leu?Leu?Trp?Thr

5 10 15 20

cct?tcc?act?gga?ctg?aag?aca?ctc?aat?ttg?gga?agc?tgt?gtg?atc?gcc 152

Pro?Ser?Thr?Gly?Leu?Lys?Thr?Leu?Asn?Leu?Gly?Ser?Cys?Val?Ile?Ala

25 20 35

aca?aac?ctt?cag?gaa?ata?cga?aat?gga?ttt?tct?gac?ata?cgg?ggc?agt 200

Thr?Asn?Leu?Gln?Glu?Ile?Arg?Asn?Gly?Phe?Ser?Asp?Ile?Arg?Gly?Ser

40 45 50

gtg?caa?gcc?aaa?gat?gga?aac?att?gac?atc?aga?atc?tta?agg?agg?act 248

Val?Gln?Ala?Lys?Asp?Gly?Asn?Ile?Asp?Ile?Arg?Ile?Leu?Arg?Arg?Thr

55 60 65

gag?tct?ttg?caa?gac?aca?aag?cct?gcg?aat?cga?tgc?tgc?ctc?ctg?cgc 296

Glu?Ser?Leu?Gln?Asp?Thr?Lys?Pro?Ala?Asn?Arg?Cys?Cys?Leu?Leu?Arg

70 75 80

cat?ttg?cta?aga?ctc?tat?ctg?gac?agg?gta?ttt?aaa?aac?tac?cag?acc 344

His?Leu?Leu?Arg?Leu?Tyr?Leu?Asp?Arg?Val?Phe?Lys?Asn?Tyr?Gln?Thr

85 90 95 100

cct?gac?cat?tat?act?ctc?cgg?aag?atc?agc?agc?ctc?gcc?aat?tcc?ttt 392

Pro?Asp?His?Tyr?Thr?Leu?Arg?Lys?Ile?Ser?Ser?Leu?Ala?Asn?Ser?Phe

105 110 115

ctt?acc?atc?aag?aag?gac?ctc?cgg?ctc?tgt?ctg?gaa?cct?cag?gca?gca 440

Leu?Thr?Ile?Lys?Lys?Asp?Leu?Arg?Leu?Cys?Leu?Glu?Pro?Gln?Ala?Ala

120 125 130

gtt?gtg?aag?gct?ttg?ggg?gaa?cta?gac?att?ctt?ctg?caa?tgg?atg?gag 488

Val?Val?Lys?Ala?Leu?Gly?Glu?Leu?Asp?Ile?Leu?Leu?Gln?Trp?Met?Glu

125 140 145

gag?aca?gaa?taggaggaaa?gtgatgctgc?tgctaagaat?attcgaggtc 537

Glu?Thr?Glu

150

aagagctcca?gtcttcaata?cctgcagagg?aggcatgacc?ccaaaccacc?atctctttac 597

tgtactagtc?ttgtgctggt?cacagtgtat?cttatttatg?cattacttgc?ttccttgcat 657

gattgtcttt?atgcatcccc?aatcttaatt?gagaccatac?ttgtataaga?tttttgtaat 717

atctttctgc?tattggatat?atttattagt?taatatattt?atttattttt?tgctattaat 777

gtatttaatt?ttttac 793

<210>4

<211>151

<212>PRT

＜213〉people

<400>4

Met?Lys?Ala?Ser?Ser?Leu?Ala?Phe?Ser?Leu?Leu?Ser?Ala?Ala?Phe?Tyr

1 5 10 15

Leu?Leu?Trp?Thr?Pro?Ser?Thr?Gly?Leu?Lys?Thr?Leu?Asn?Leu?Gly?Ser

20 25 30

Cys?Val?Ile?Ala?Thr?Asn?Leu?Gln?Glu?Ile?Arg?Asn?Gly?Phe?Ser?Asp

35 40 45

Ile?Arg?Gly?Ser?Val?Gln?Ala?Lys?Asp?Gly?Asn?Ile?Asp?Ile?Arg?Ile

50 55 60

Leu?Arg?Arg?Thr?Glu?Ser?Leu?Gln?Asp?Thr?Lys?Pro?Ala?Asn?Arg?Cys

65 70 75 80

Cys?Leu?Leu?Arg?His?Leu?Leu?Arg?Leu?Tyr?Leu?Asp?Arg?Val?Phe?Lys

85 90 95

Asn?Tyr?Gln?Thr?Pro?Asp?His?Tyr?Thr?Leu?Arg?Lys?Ile?Ser?Ser?Leu

100 105 110

Ala?Asn?Ser?Phe?Leu?Thr?Ile?Lys?Lys?Asp?Leu?Arg?Leu?Cys?Leu?Glu

115 120 125

Pro?Gln?Ala?Ala?Val?Val?Lys?Ala?Leu?Gly?Glu?Leu?Asp?Ile?Leu?Leu

130 135 140

Gln?Trp?Met?Glu?Glu?Thr?Glu

145 150

<210>5

<211>253

<212>DNA

＜213〉people

<400>5

ctttgaattc?ctagctcctg?tggtctccag?atttcaggcc?taagatgaaa?gcctctagtc 60

ttgccttcag?ccttctctct?gctgcgtttt?atctcctatg?gactccttcc?actggactga 120

agacactcaa?tttgggaagc?tgtgtgatcg?ccacaaacct?tcaggaaata?cgaaatggat 180

tttctgagat?acggggcagt?gtgcaagcca?aagatggaaa?cattgacatc?agaatcttaa 240

ggaggactga?gtc 253

<210>6

<211>24

<212>DNA

＜213〉people

<400>6

attcctagct?cctgtggtct?ccag 24

<210>7

<211>25

<212>DNA

＜213〉people

<400>7

ctctgctgcg?ttttatctcc?tatgg 25

<210>8

<211>22

<212>DNA

＜213〉people

<400>8

tcccaaattg?agtgtcttca?gt 22

<210>9

<211>45

<212>DNA

＜213〉people

<400>9

cacagcttcc?caaattgagt?gtcttcagtc?cagtggaagg?agtcc 45

<210>10

<211>747

<212>DNA

＜213〉people

<400>10

ttttctgaca?tacggggcag?tgtgcaagcc?aaagatggaa?acattgacat?cagaatctta 60

aggaggactg?agtctttgca?agacacaaag?cctgcgaatc?gatgctgcct?cctgcgccat 120

ttgctaagac?tctatctgga?cagggtattt?aaaaactacc?agacccctga?ccattatact 180

ctccggaaga?tcagcagcct?cgccaattcc?tttcttacca?tcaagaagga?cctcc9gctc 240

tgtcatgccc?acatgacatg?ccattgtggg?gaggaagcaa?tgaagaaata?cagccagatt 300

ctgagtcact?ttgaaaagct?ggaacctcag?gcagcagttg?tgaaggcttt?gggggaacta 360

gacattcttc?tgcaatggat?ggaggagaca?gaataggagg?aaagtgatgc?tgctgctaag 420

aatattcgag?gtcaagagct?ccagtcttca?atacctgcag?aggaggcatg?accccaaacc 480

accatctctt?tactgtacta?gtcttgtgct?ggtcacagtg?tatcttattt?atgcattact 540

tgcttccttg?catgattgtc?tttatgcatc?cccaatctta?attgagacca?tacttgtata 600

agatttttgt?aatatctttc?tgctattgga?tatatttatt?agttaatata?tttatttatt 660

ttttgctatt?aatgtattta?attttttact?tgggcatgaa?actttaaaaa?aaattcacaa 720

gattatattt?ataacctgac?tagagca 747

<210>11

<211>614

<212>DNA

＜213〉people

<400>11

ttttctgaca?tacggggcag?tgtgcaagcc?aaagatggaa?acattgacat?cagaatctta 60

aggaggactg?agtctttgca?agacacaaag?cctgcgaatc?gatgctgcct?cctgcgccat 120

ttgctaagac?tctatctgga?cagggtattt?aaaaactacc?agacccctga?ccattatact 180

ctccggaaga?tcagcagcct?cgccaattcc?tttcttacca?tcaagaagga?cctccggctc 240

tgtctggaac?ctcaggcagc?agttgtgaag?gctttgg9gg?aactagacat?tcttctgcaa 300

tggatggagg?agacagaata?ggaggaaagt?gatgctgctg?ctaagaatat?tcgaggtcaa 360

gagctccagt?cttcaatacc?tgcagaggag?gcatgacccc?aaaccaccat?ctctttactg 420

tactagtctt?gtgctggtca?cagtgtatct?tatttatgca?ttacttgctt?ccttgcatga 480

ttgtctttat?gcatccccaa?tcttaattga?gaccatactt?gtataagatt?tttgtaatat 540

ctttctgcta?ttggatatat?ttattagtta?atatatttat?ttattttttg?ctattaatgt 600

atttaatttt?ttac 614

<210>12

<211>152

<212>PRT

＜213〉people

<400>12

Leu?Lys?Thr?Leu?Asn?Leu?Gly?Ser?Cys?Val?Ile?Ala?Thr?Asn?Leu?Gln

1 5 10 15

Glu?Ile?Arg?Asn?Gly?Phe?Ser?Asp?Ile?Arg?Gly?Ser?Val?Gln?Ala?Lys

20 25 30

Asp?Gly?Asn?Ile?Asp?Ile?Arg?Ile?Leu?Arg?Arg?Thr?Glu?Ser?Leu?Gln

35 40 45

Asp?Thr?Lys?Pro?Ala?Asn?Arg?Cys?Cys?Leu?Leu?Arg?His?Leu?Leu?Arg

50 55 60

Leu?Tyr?Leu?Asp?Arg?Val?Phe?Lys?Asn?Tyr?Gln?Thr?Pro?Asp?His?Tyr

65 70 75 80

Thr?Leu?Arg?Lys?Ile?Ser?Ser?Leu?Ala?Asn?Ser?Phe?Leu?Thr?Ile?Lys

85 90 95

Lys?Asp?Leu?Arg?Leu?Cys?His?Ala?His?Met?Thr?Cys?His?Cys?Gly?Glu

100 105 110

Glu?Ala?Met?Lys?Lys?Tyr?Ser?Gln?Ile?Leu?Ser?His?Phe?Glu?Lys?Leu

115 120 125

Glu?Pro?Gln?Ala?Ala?Val?Val?Lys?Ala?Leu?Gly?Glu?Leu?Asp?Ile?Leu

130 135 140

Leu?Gln?Trp?Met?Glu?Glu?Thr?Glu

145 150

<210>13

<211>127

<212>PRT

＜213〉people

<400>13

Leu?Lys?Thr?Leu?Asn?Leu?Gly?Ser?Cys?Val?Ile?Ala?Thr?Asn?Leu?Gln

1 5 10 15

Glu?Ile?Arg?Asn?Gly?Phe?Ser?Asp?Ile?Arg?Gly?Ser?Val?Gln?Ala?Lys

20 25 30

Asp?Gly?Asn?Ile?Asp?Ile?Arg?Ile?Leu?Arg?Arg?Thr?Glu?Ser?Leu?Gln

35 40 45

Asp?Thr?Lys?Pro?Ala?Asn?Arg?Cys?Cys?Leu?Leu?Arg?His?Leu?Leu?Arg

50 55 60

Leu?Tyr?Leu?Asp?Arg?Val?Phe?Lys?Asn?Tyr?Gln?Thr?Pro?Asp?His?Tyr

65 70 75 80

Thr?Leu?Arg?Lys?Ile?Ser?Ser?Leu?Ala?Asn?Ser?Phe?Leu?Thr?Ile?Lys

85 90 95

Lys?Asp?Leu?Arg?Leu?Cys?Leu?Glu?Pro?Gln?Ala?Ala?Val?Val?Lys?Ala

100 105 110

Leu?Gly?Glu?Leu?Asp?Ile?Leu?Leu?Gln?Trp?Met?Glu?Glu?Thr?Glu

115 120 125

<210>14

<211>15

<212>PRT

＜213〉people

<400>14

Ile?Ala?Thr?Asn?Leu?Gln?Glu?Ile?Arg?Asn?Gly?Phe?Ser?Asp?Ile

1 5 10 15

<210>15

<211>15

<212>PRT

＜213〉people

<400>15

Leu?Asp?Arg?Val?Phe?Lys?Asn?Tyr?Gln?Thr?Pro?Asp?His?Tyr?Thr

1 5 10 15

<210>16

<211>15

<212>PRT

＜213〉people

<400>16

Leu?Ala?Asn?Ser?Phe?Leu?Thr?Ile?Lys?Lys?Asp?Leu?Arg?Leu?Cys

1 5 10 15

<210>17

<211>15

<212>PRT

＜213〉people

<400>17

Val?Val?Lys?Ala?Leu?Gly?Glu?Leu?Asp?Ile?Leu?Leu?Gln?Trp?Met

1 5 10 15

<210>18

<211>824

<212>DNA

<213>Mus?musculus

<220>

<221>CDS

<222>(71)...(598)

<400>18

tgggagacat?cgatagccct?gattgatctc?tttgaattt?cgcttctggt?ctccaggatc 60

taggtgtaag?atg?aaa?ggc?ttt?ggt?ctt?gcc?ttt?gga?ctg?ttc?tcc?gct 109

Met?Lys?Gly?Phe?Gly?Leu?Ala?Phe?Gly?Leu?Phe?Ser?Ala

1 5 10

gtg?ggt?ttt?ctt?ctc?tgg?act?cct?tta?act?ggg?ctc?aag?acc?ctc?cat 157

Val?Gly?Phe?Leu?Leu?Trp?Thr?Pro?Leu?Thr?Gly?Leu?Lys?Thr?Leu?His

15 20 25

ttg?gga?agc?tgt?gtg?att?act?gca?aac?cta?cag?gca?ata?caa?aag?gaa 205

Leu?Gly?Ser?Cys?Val?Ile?Thr?Ala?Asn?Leu?Gln?Ala?Ile?Gln?Lys?Glu

30 35 40 45

ttt?tct?gag?att?cgg?gat?agt?gtg?caa?gct?gaa?gat?aca?aat?att?gac 253

Phe?Ser?Glu?Ile?Arg?Asp?Ser?Val?Gln?Ala?Glu?Asp?Thr?Asn?Ile?Asp

50 55 60

atc?aga?att?tta?agg?acg?act?gag?tct?ttg?aaa?gac?ata?aag?tct?ttg 301

Ile?Arg?Ile?Leu?Arg?Thr?Thr?Glu?Ser?Leu?Lys?Asp?Ile?Lys?Ser?Leu

65 70 75

gat?agg?tgc?tgc?ttc?ctt?cgt?cat?cta?gtg?aga?ttc?tat?ctg?gac?agg 349

Asp?Arg?Cys?Cys?Phe?Leu?Arg?His?Leu?Val?Arg?Phe?Tyr?Leu?Asp?Arg

80 85 90

gta?ttc?aaa?gtc?tac?cag?acc?cct?gac?cac?cat?acc?ctg?aga?aag?atc 397

Val?Phe?Lys?Val?Tyr?Gln?Thr?Pro?Asp?His?His?Thr?Leu?Arg?Lys?Ile

95 100 105

agc?agc?ctc?gcc?aac?tcc?ttt?ctt?atc?atc?aag?aag?gac?ctc?tca?gtc 445

Ser?Ser?Leu?Ala?Asn?Ser?Phe?Leu?Ile?Ile?Lys?Lys?Asp?Leu?Ser?Val

110 115 120 125

tgt?cat?tct?cac?atg?gca?tgt?cat?tgt?ggg?gaa?gaa?gca?atg?gag?aaa 493

Cys?His?Ser?His?Met?Ala?Cys?His?Cys?Gly?Glu?Glu?Ala?Met?Glu?Lys

130 135 140

tac?aac?caa?att?ctg?agt?cac?ttc?ata?gag?ttg?gaa?ctt?cag?gca?gcg 541

Tyr?Asn?Gln?Ile?Leu?Ser?His?Phe?Ile?Glu?Leu?Glu?Leu?Gln?Ala?Ala

145 150 155

gtg?gta?aag?gct?ttg?gga?gaa?cta?ggc?att?ctt?ctg?aga?tgg?atg?gag 589

Val?Val?Lys?Ala?Leu?Gly?Glu?Leu?Gly?Ile?Leu?Leu?Arg?Trp?Met?Glu

160 165 170

gag?atg?cta?tagatgaaag?tggagaggct?gctgagaaca?ctcctgtcca 638

Glu?Met?Leu

175

agaatctcag?acctcagcac?catgaagaca?tggccccagg?tgctggcatt?tctactcaag 698

agttccagtc?ctcagcacca?cgaagatggc?ctcaaaccac?cacccctttg?tgatataact 758

tagtgctagc?tatgtgtata?ttatttctac?attattggct?cccttatgtg?aatgccttca 818

tgtgtc 824

<210>19

<211>176

<212>PRT

<213>Mus?musculus

<400>19

Met?Lys?Gly?Phe?Gly?Leu?Ala?Phe?Gly?Leu?Phe?Ser?Ala?Val?Gly?Phe

1 5 10 15

Leu?Leu?Trp?Thr?Pro?Leu?Thr?Gly?Leu?Lys?Thr?Leu?His?Leu?Gly?Ser

20 25 30

Cys?Val?Ile?Thr?Ala?Asn?Leu?Gln?Ala?Ile?Gln?Lys?Glu?Phe?Ser?Glu

35 40 45

Ile?Arg?Asp?Ser?Val?Gln?Ala?Glu?Asp?Thr?Asn?Ile?Asp?Ile?Arg?Ile

50 55 60

Leu?Arg?Thr?Thr?Glu?Ser?Leu?Lys?Asp?Ile?Lys?Ser?Leu?Asp?Arg?Cys

65 70 75 80

Cys?Phe?Leu?Arg?His?Leu?Val?Arg?Phe?Tyr?Leu?Asp?Arg?Val?Phe?Lys

85 90 95

Val?Tyr?Gln?Thr?Pro?Asp?His?His?Thr?Leu?Arg?Lys?Ile?Ser?Ser?Leu

100 105 110

Ala?Asn?Ser?Phe?Leu?Ile?Ile?Lys?Lys?Asp?Leu?Ser?Val?Cys?His?Ser

115 120 125

His?Met?Ala?Cys?His?Cys?Gly?Glu?Glu?Ala?Met?Glu?Lys?Tyr?Asn?Gln

130 135 140

Ile?Leu?Ser?His?Phe?Ile?Glu?Leu?Glu?Leu?Gln?Ala?Ala?Val?Val?Lys

145 150 155 160

Ala?Leu?Gly?Glu?Leu?Gly?Ile?Leu?Leu?Arg?Trp?Met?Glu?Glu?Met?Leu

165 170 175

<210>20

<211>152

<212>PRT

<213>Mus?musculus

<400>20

Leu?Lys?Thr?Leu?His?Leu?Gly?Ser?Cys?Val?Ile?Thr?Ala?Asn?Leu?Gln

1 5 10 15

Ala?Ile?Gln?Lys?Glu?Phe?Ser?Glu?Ile?Arg?Asp?Ser?Val?Gln?Ala?Glu

20 25 30

Asp?Thr?Asn?Ile?Asp?Ile?Arg?Ile?Leu?Arg?Thr?Thr?Glu?Ser?Leu?Lys

35 40 45

Asp?Ile?Lys?Ser?Leu?Asp?Arg?Cys?Cys?Phe?Leu?Arg?His?Leu?Val?Arg

50 55 60

Phe?Tyr?Leu?Asp?Arg?Val?Phe?Lys?Val?Tyr?Gln?Thr?Pro?Asp?His?His

65 70 75 80

Thr?Leu?Arg?Lys?Ile?Ser?Ser?Leu?Ala?Asn?Ser?Phe?Leu?Ile?Ile?Lys

85 90 95

Lys?Asp?Leu?Ser?Val?Cys?His?Ser?His?Met?Ala?Cys?His?Cys?Gly?Glu

100 105 110

Glu?Ala?Met?Glu?Lys?Tyr?Ash?Gln?Ile?Leu?Ser?His?Phe?Ile?Glu?Leu

115 120 125

Glu?Leu?Gln?Ala?Ala?Val?Val?Lys?Ala?Leu?Gly?Glu?Leu?Gly?Ile?Leu

130 135 140

Leu?Arg?Trp?Met?Glu?Glu?Met?Leu

145 150

<210>21

<211>16

<212>PRT

<213>Mus?musculus

<400>21

Ile?Thr?Ala?Asn?Leu?Gln?Ala?Ile?Gln?Lys?Glu?Phe?Ser?Glu?Ile?Arg

1 5 10 15

<210>22

<211>15

<212>PRT

<213>Mus?musculus

<400>22

Leu?Asp?Arg?Val?Phe?Lys?Val?Tyr?Gln?Thr?Pro?Asp?His?His?Thr

1 5 10 15

<210>23

<211>15

<212>PRT

<213>Mus?musculus

<400>23

Leu?Ala?Asn?Ser?Phe?Leu?Ile?Ile?Lys?Lys?Asp?Leu?Ser?Val?Cys

1 5 10 15

<210>24

<211>15

<212>PRT

<213>Mus?muculus

<400>24

Val?Val?Lys?Ala?Leu?Gly?Glu?Leu?Gly?Ile?Leu?Leu?Arg?Trp?Met

1 5 10 15

<210>25

<211>144

<212>PRT

<213>Mus?muculus

<400>25

Cys?Val?Ile?Thr?Ala?Asn?Leu?Gln?Ala?Ile?Gln?Lys?Glu?Phe?Ser?Glu

1 5 10 15

Ile?Arg?Asp?Ser?Val?Gln?Ala?Glu?Asp?Thr?Asn?Ile?Asp?Ile?Arg?Ile

20 25 30

Leu?Arg?Thr?Thr?Glu?Ser?Leu?Lys?Asp?Ile?Lys?Ser?Leu?Asp?Arg?Cys

35 40 45

Cys?Phe?Leu?Arg?His?Leu?Val?Arg?Phe?Tyr?Leu?Asp?Arg?Val?Phe?Lys

50 55 60

Val?Tyr?Gln?Thr?Pro?Asp?His?His?Thr?Leu?Arg?Lys?Ile?Ser?Ser?Leu

65 70 75 80

Ala?Asn?Ser?Phe?Leu?Ile?Ile?Lys?Lys?Asp?Leu?Ser?Val?Cys?His?Ser

85 90 95

His?Met?Ala?Cys?His?Cys?Gly?Glu?Glu?Ala?Met?Glu?Lys?Tyr?Asn?Gln

100 105 110

Ile?Leu?Ser?His?Phe?Ile?Glu?Leu?Glu?Leu?Gln?Ala?Ala?Val?Val?Lys

115 120 125

Ala?Leu?Gly?Glu?Leu?Gly?Ile?Leu?Leu?Arg?Trp?Met?Glu?Glu?Met?Leu

130 135 140

<210>26

<211>144

<212>PRT

＜213〉people

<400>26

Cys?Val?Ile?Ala?Thr?Asn?Leu?Gln?Glu?Ile?Arg?Asn?Gly?Phe?Ser?Asp

1 5 10 15

Ile?Arg?Gly?Ser?Val?Gln?Ala?Lys?Asp?Gly?Asn?Ile?Asp?Ile?Arg?Ile

20 25 30

Leu?Arg?Arg?Thr?Glu?Ser?Leu?Gln?Asp?Thr?Lys?Pro?Ala?Asn?Arg?Cys

35 40 45

Cys?Leu?Leu?Arg?His?Leu?Leu?Arg?Leu?Tyr?Leu?Asp?Arg?Val?Phe?Lys

50 55 60

Asn?Tyr?Gln?Thr?Pro?Asp?His?Tyr?Thr?Leu?Arg?Lys?Ile?Ser?Ser?Leu

65 70 75 80

Ala?Asn?Ser?Phe?Leu?Thr?Ile?Lys?Lys?Asp?Leu?Arg?Leu?Cys?His?Ala

85 90 95

His?Met?Thr?Cys?His?Cys?Gly?Glu?Glu?Ala?Met?Lys?Lys?Tyr?Ser?Gln

100 105 110

Ile?Leu?Ser?His?Phe?Glu?Lys?Leu?Glu?Pro?Gln?Ala?Ala?Val?Val?Lys

115 120 125

Ala?Leu?Gly?Glu?Leu?Asp?Ile?Leu?Leu?Gln?Trp?Met?Glu?Glu?Thr?Glu

130 135 140

<210>27

<211>38

<212>PRT

＜213〉people

<400>27

Cys?Gly?Glu?Glu?Ala?Met?Lys?Lys?Tyr?Ser?Gln?Ile?Leu?Ser?His?Phe

1 5 10 15

Glu?Lys?Leu?Glu?Pro?Gln?Ala?Ala?Val?Val?Lys?Ala?Leu?Gly?Glu?Leu

20 25 30

Asp?Ile?Leu?Leu?Gln?Trp

35

<210>28

<211>71

<212>PRT

＜213〉people

<400>28

Ile?Ala?Thr?Asn?Leu?Gln?Glu?Ile?Arg?Asn?Gly?Phe?Ser?Asp?Ile?Arg

1 5 10 15

Gly?Ser?Val?Gln?Ala?Lys?Asp?Gly?Asn?Ile?Asp?Ile?Arg?Ile?Leu?Arg

20 25 30

Arg?Thr?Glu?Ser?Leu?Gln?Asp?Thr?Lys?Pro?Ala?Asn?Arg?Cys?Cys?Leu

35 40 45

Leu?Arg?His?Leu?Leu?Arg?Leu?Tyr?Leu?Asp?Arg?Val?Phe?Lys?Asn?Tyr

50 55 60

Gln?Thr?Pro?Asp?His?Tyr?Thr

65 70

<210>29

<211>92

<212>PRT

＜213〉people

<400>29

Ile?Ala?Thr?Asn?Leu?Gln?Glu?Ile?Arg?Asn?Gly?Phe?Ser?Asp?Ile?Arg

1 5 10 15

Gly?Ser?Val?Gln?Ala?Lys?Asp?Gly?Asn?Ile?Asp?Ile?Arg?Ile?Leu?Arg

20 25 30

Arg?Thr?Glu?Ser?Leu?Gln?Asp?Thr?Lys?Pro?Ala?Asn?Arg?Cys?Cys?Leu

35 40 45

Leu?Arg?His?Leu?Leu?Arg?Leu?Tyr?Leu?Asp?Arg?Val?Phe?Lys?Asn?Tyr

50 55 60

Gln?Thr?Pro?Asp?His?Tyr?Thr?Leu?Arg?Lys?Ile?Ser?Ser?Leu?Ala?Asn

65 70 75 80

Ser?Phe?Leu?Thr?Ile?Lys?Lys?Asp?Leu?Arg?Leu?Cys

85 90

<210>30

<211>82

<212>PRT

＜213〉people

<400>30

Leu?Asp?Arg?Val?Phe?Lys?Asn?Tyr?Gln?Thr?Pro?Asp?His?Tyr?Thr?Leu

1 5 10 15

Arg?Lys?Ile?Ser?Ser?Leu?Ala?Asn?Ser?Phe?Leu?Thr?Ile?Lys?Lys?Asp

20 25 30

Leu?Arg?Leu?Cys?His?Ala?His?Met?Thr?Cys?His?Cys?Gly?Glu?Glu?Ala

35 40 45

Met?Lys?Lys?Tyr?Ser?Gln?Ile?Leu?Ser?His?Phe?Glu?Lys?Leu?Glu?Pro

50 55 60

Gln?Ala?Ala?Val?Val?Lys?Ala?Leu?Gly?Glu?Leu?Asp?Ile?Leu?Leu?Gln

65 70 75 80

Trp?Met

<210>31

<211>36

<212>PRT

＜213〉people

<400>31

Leu?Asp?Arg?Val?Phe?Lys?Asn?Tyr?Gln?Thr?Pro?Asp?His?Tyr?Thr?Leu

1 5 10 15

Arg?Lys?Ile?Ser?Ser?Leu?Ala?Asn?Ser?Phe?Leu?Thr?Ile?Lys?Lys?Asp

20 25 30

Leu?Arg?Leu?Cys

35

<210>32

<211>61

<212>PRT

＜213〉people

<400>32

Leu?Ala?Asn?Ser?Phe?Leu?Thr?Ile?Lys?Lys?Asp?Leu?Arg?Leu?Cys?His

1 5 10 15

Ala?His?Met?Thr?Cys?His?Cys?Gly?Glu?Glu?Ala?Met?Lys?Lys?Tyr?Ser

20 25 30

Gln?Ile?Leu?Ser?His?Phe?Glu?Lys?Leu?Glu?Pro?Gln?Ala?Ala?Val?Val

35 40 45

Lys?Ala?Leu?Gly?Glu?Leu?Asp?Ile?Leu?Leu?Gln?Trp?Met

50 55 60

<210>33

<211>756

<212>DNA

<213>Mus?musculus

<220>

<221>CDS

<222>(71)...(532)

<400>33

tgggagacat?cgatagccct?gattgatctc?tttgaatttt?cgcttctggt?ctccaggatc 60

taggtgtaag?atg?aaa?ggc?ttt?ggt?ctt?gcc?ttt?gga?ctg?ttc?tcc?gct 109

Met?Lys?Gly?Phe?Gly?Leu?Ala?Phe?Gly?Leu?Phe?Ser?Ala

1 5 10

gtg?ggt?ttt?ctt?ctc?tgg?act?cct?tta?act?ggg?ctc?aag?acc?ctc?cat 157

Val?Gly?Phe?Leu?Leu?Trp?Thr?Pro?Leu?Thr?Gly?Leu?Lys?Thr?Leu?His

15 20 25

ttg?gga?agc?tgt?gtg?att?act?gca?aac?cta?cag?gca?ata?caa?aag?gaa 205

Leu?Gly?Ser?Cys?Val?Ile?Thr?Ala?Asn?Leu?Gln?Ala?Ile?Gln?Lys?Glu

30 35 40 45

ttt?tct?gag?att?cgg?gat?agt?gtg?tct?ttg?gat?agg?tgc?tgc?ttc?ctt 253

Phe?Ser?Glu?Ile?Arg?Asp?Ser?Val?Ser?Leu?Asp?Arg?Cys?Cys?Phe?Leu

50 55 60

cgt?cat?cta?gtg?aga?ttc?tat?ctg?gac?agg?gta?ttc?aaa?gtc?tac?cag 301

Arg?His?Leu?Val?Arg?Phe?Tyr?Leu?Asp?Arg?Val?Phe?Lys?Val?Tyr?Gln

65 70 75

acc?cct?gac?cac?cat?acc?ctg?aga?aag?atc?agc?agc?ctc?gcc?aac?tcc 349

Thr?Pro?Asp?His?His?Thr?Leu?Arg?Lys?Ile?Ser?Ser?Leu?Ala?Asn?Ser

80 85 90

ttt?ctt?atc?atc?aag?aag?gac?ctc?tca?gtc?tgt?cat?tct?cac?atg?gca 397

Phe?Leu?Ile?Ile?Lys?Lys?Asp?Leu?Ser?Val?Cys?His?Ser?His?Met?Ala

95 100 105

tgt?cat?tgt?ggg?gaa?gaa?gca?atg?gag?aaa?tac?aac?caa?att?ctg?agt 445

Cys?His?Cys?Gly?Glu?Glu?Ala?Met?Glu?Lys?Tyr?Asn?Gln?Ile?Leu?Ser

110 115 120 125

cac?ttc?ata?gag?ttg?gaa?ctt?cag?gca?gcg?gtg?gta?aag?gct?ttg?gga 493

His?Phe?Ile?Glu?Leu?Glu?Leu?Gln?Ala?Ala?Val?Val?Lys?Ala?Leu?Gly

130 135 140

gaa?cta?ggc?att?ctt?ctg?aga?tgg?atg?gag?gag?atg?cta?tagatgaaag 542

Glu?Leu?Gly?Ile?Leu?Leu?Arg?Trp?Met?Glu?Glu?Met?Leu

145 150

tggataggct?gctgagaaca?ctcctgtcca?agaatctcag?acctcagcac?catgaagaca 602

tggccccagg?tgctggcatt?tctactcaag?agttccagtc?ctcagcacca?cgaagatggc 662

ctcaaaccac?cacccctttg?tgatataact?tagtgctagc?tatgtgtata?ttatttctac 722

attattggct?cccttatgtg?aatgccttca?tgtg 756

<210>34

<211>154

<212>PRT

<213>Mus?musculus

<400>34

Met?Lys?Gly?Phe?Gly?Leu?Ala?Phe?Gly?Leu?Phe?Ser?Ala?Val?Gly?Phe

1 5 10 15

Leu?Leu?Trp?Thr?Pro?Leu?Thr?Gly?Leu?Lys?Thr?Leu?His?Leu?Gly?Ser

20 25 30

Cys?Val?Ile?Thr?Ala?Asn?Leu?Gln?Ala?Ile?Gln?Lys?Glu?Phe?Ser?Glu

35 40 45

Ile?Arg?Asp?Ser?Val?Ser?Leu?Asp?Arg?Cys?Cys?Phe?Leu?Arg?His?Leu

50 55 60

Val?Arg?Phe?Tyr?Leu?Asp?Arg?Val?Phe?Lys?Val?Tyr?Gln?Thr?Pro?Asp

65 70 75 80

His?His?Thr?Leu?Arg?Lys?Ile?Ser?Ser?Leu?Ala?Asn?Ser?Phe?Leu?Ile

85 90 95

Ile?Lys?Lys?Asp?Leu?Ser?Val?Cys?His?Ser?His?Met?Ala?Cys?His?Cys

100 105 110

Gly?Glu?Glu?Ala?Met?Glu?Lys?Tyr?Asn?Gln?Ile?Leu?Ser?His?Phe?Ile

115 120 125

Glu?Leu?Glu?Leu?Gln?Ala?Ala?Val?Val?Lys?Ala?Leu?Gly?Glu?Leu?Gly

130 135 140

Ile?Leu?Leu?Arg?Trp?Met?Glu?Glu?Met?Leu

145 150

<210>35

<211>130

<212>PRT

<213>Mus?musculus

<400>35

Leu?Lys?Thr?Leu?His?Leu?Gly?Ser?Cys?Val?Ile?Thr?Ala?Asn?Leu?Gln

1 5 10 15

Ala?Ile?Gln?Lys?Glu?Phe?Ser?Glu?Ile?Arg?Asp?Ser?Val?Ser?Leu?Asp

20 25 30

Arg?Cys?Cys?Phe?Leu?Arg?His?Leu?Val?Arg?Phe?Tyr?Leu?Asp?Arg?Val

35 40 45

Phe?Lys?Val?Tyr?Gln?Thr?Pro?Asp?His?His?Thr?Leu?Arg?Lys?Ile?Ser

50 55 60

Ser?Leu?Ala?Asn?Ser?Phe?Leu?Ile?Ile?Lys?Lys?Asp?Leu?Ser?Val?Cys

65 70 75 80

His?Ser?His?Met?Ala?Cys?His?Cys?Gly?Glu?Glu?Ala?Met?Glu?Lys?Tyr

85 90 95

Asn?Gln?Ile?Leu?Ser?His?Phe?Ile?Glu?Leu?Glu?Leu?Gln?Ala?Ala?Val

100 105 110

Val?Lys?Ala?Leu?Gly?Glu?Leu?Gly?Ile?Leu?Leu?Arg?Trp?Met?Glu?Glu

115 120 125

Met?Leu

130

<210>36

<211>27

<212>DNA

＜213〉people

<400>36

agattctatc?tggacagggt?attcaaa 27

<210>37

<211>17

<212>DNA

＜213〉people

<400>37

gcgaggctga?tctttct 17

<210>38

<211>25

<212>DNA

<213>Mus?musculis

<400>38

tggcgaggct?gctgatcttt?ctcag 25

<210>39

<211>25

<212>DNA

<213>Mus?musculis

<400>39

ctttatgtct?ttcaaagact?cagtc 25

<210>40

<211>26

<212>DNA

<213>Mus?musculis

<400>40

catcagaatt?ttaaggacga?ctgagt 26

<210>41

<211>25

<212>DNA

<213>Mus?musculis

<400>41

ggtggtcagg?ggtctggtag?acttt 25

<210>42

<211>23

<212>DNA

<213>Mus?musculis

<400>42

ggtgcatatt?cctggtggct?aga 23

<210>43

<211>25

<212>DNA

<213>Mus?musculis

<400>43

attgcagtgt?aagggaatac?agaga 25

Claims

1. it is at least 90% identical that the separation polynucleotide of coded polypeptide, said polypeptide and the polypeptide of SEQ ID No:4 or SEQ ID No:13 have.

2. the separation polynucleotide of claim 1, wherein said polynucleotide encoding contains the polypeptide of the aminoacid sequence of SEQ ID No:4 or SEQ ID No:13.

3. the separation polynucleotide of claim 1, wherein said polynucleotide are SEQ IDNo:3.

4. the isolated polypeptide identical with being selected from SEQ ID No:4 or SEQ ID No:13 polypeptide at least 90%.

5. the isolated polypeptide of claim 4, wherein said polypeptide is selected from SEQ ID No:4 and SEQ ID No:13.

6. optionally in conjunction with the antibody of polypeptide epitope part, described polypeptide is selected from: SEQ IDNo:4 and SEQ ID No:13.

Can with the antiidiotypic antibody of the antibodies of claim 6.